Antibody molecules that bind to IL-6 receptor

ABSTRACT

The present invention provides pharmaceutical compositions comprising second-generation molecules that are superior than TOCILIZUMAB, by altering the amino acid sequences of the variable and constant regions of TOCILIZUMAB, which is a humanized anti-IL-6 receptor IgG1 antibody, to enhance the antigen-neutralizing ability and increase the pharmacokinetics, so that the therapeutic effect is exerted with a less frequency of administration, and the immunogenicity, safety and physicochemical properties (stability and homogeneity) are improved. The present invention also provides methods for producing these pharmaceutical compositions. The present inventors have successfully generated second-generation molecules that are superior to TOCILIZUMAB by appropriately combining amino acid sequence alterations in the CDR domains, variable regions, and constant regions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the National Stage of International ApplicationSerial No. PCT/JP2009/066590, filed on Sep. 25, 2009, which claims thebenefit of Japanese Application Serial Nos. 2009-067925, filed on Mar.19, 2009; 2009-060806, filed on Mar. 13, 2009; and 2008-248213, filed onSep. 26, 2008. The contents of the foregoing applications areincorporated by reference herein.

TECHNICAL FIELD

The present invention relates to pharmaceutical compositions comprisingan anti-IL-6 receptor antibody as an active ingredient, methods forproducing the compositions, and such.

BACKGROUND ART

Antibodies are drawing attention as pharmaceuticals as they are highlystable in plasma and have few adverse effects. Among them, a number ofIgG-type antibody pharmaceuticals are available on the market and manyantibody pharmaceuticals are currently under development (Non-PatentDocuments 1 and 2). IL-6 is a cytokine involved in various autoimmunediseases, inflammatory diseases, malignant tumors, and so on (Non-PatentDocument 3). TOCILIZUMAB, a humanized anti-IL-6 receptor IgG1 antibody,specifically binds to the IL-6 receptor. It is thought that TOCILIZUMABcan be used as a therapeutic agent for IL-6-associated diseases such asrheumatoid arthritis, since it neutralizes the biological activity ofIL-6 (Patent Documents 1 to 3, and Non-Patent Document 4). TOCILIZUMABhas been approved as a therapeutic agent for Castleman's disease andrheumatoid arthritis in Japan (Non-Patent Document 5).

Humanized antibodies such as TOCILIZUMAB are first-generation antibodypharmaceuticals. Second-generation antibody pharmaceuticals arecurrently being developed by improving the efficacy, convenience, andcost of first-generation antibody pharmaceuticals. Various technologiesthat are applicable to second-generation antibody pharmaceuticals arebeing developed. Technologies for enhancing effector function,antigen-binding ability, pharmacokinetics, and stability, as well astechnologies for reducing the risk of immunogenicity have been reported.As methods for enhancing drug efficacy or reducing dosage, technologiesthat enhance antibody-dependent cell-mediated cytotoxic activity (ADCCactivity) or complement-dependent cytotoxic activity (CDC activity)through amino acid substitution in the Fc region of an IgG antibody havebeen reported (Non-Patent Document 6). Furthermore, affinity maturationhas been reported as a technology for enhancing antigen-binding abilityor antigen-neutralizing ability (Non-Patent Document 7). This technologyenables one to enhance antigen-binding activity by introducing aminoacid mutations into the complementarity determining (CDR) region of avariable region or such. The enhancement of antigen-binding abilityimproves in vitro biological activity or reduces dosage, and furthermoreimproves in vivo efficacy (Non-Patent Document 8). Currently, clinicaltrials are being conducted to assess Motavizumab (produced by affinitymaturation), which is expected to have a superior efficacy thanPalivizumab, a first-generation anti-RSV antibody pharmaceutical(Non-Patent Document 9). An anti-IL-6 receptor antibody with an affinityof about 0.05 nM (i.e., greater affinity than that of TOCILIZUMAB) hasbeen reported (Patent Document 4). However, there is no reportdescribing a human, humanized, or chimeric antibody having an affinitygreater than 0.05 nM.

A problem encountered with current antibody pharmaceuticals is the highproduction cost associated with the administration of extremely largequantities of protein. For example, the dosage of TOCILIZUMAB, ahumanized anti-IL-6 receptor IgG1 antibody, has been estimated to beabout 8 mg/kg/month by intravenous injection (Non-Patent Document 4).Its preferred form of administration is subcutaneous formulation inchronic autoimmune diseases. In general, it is necessary thatsubcutaneous formulations are high-concentration formulations. From theperspective of stability or such, the limit for IgG-type antibodyformulations is generally about 100 mg/ml (Non-Patent Document 10).Low-cost, convenient second-generation antibody pharmaceuticals that canbe administered subcutaneously in longer intervals can be provided byincreasing the half-life of an antibody in the plasma to prolong itstherapeutic effect and thereby reduce the amount of proteinadministered, and by conferring the antibody with high stability.

FcRn is closely involved in antibody pharmacokinetics. With regard todifferences in the plasma half-life of antibody isotypes, IgG1 and IgG2are known to have superior plasma half-life than IgG3 and IgG4(Non-Patent Document 11). As a method for further improving the plasmahalf-life of IgG1 and IgG2 antibodies which have superior plasmahalf-lives, substitution of amino acids in the constant region whichenhances the binding to FcRn has been reported (Non-Patent Documents 12and 13). From the viewpoint of immunogenicity, further improvement ofthe plasma half-life is performed by substituting amino acids preferablyin the variable region rather than in the constant region (PatentDocument 5). However, there is no report to date on the improvement ofthe plasma half-life of IL-6 receptor antibodies through alteration ofthe variable region.

Another important problem encountered in the development ofbiopharmaceuticals is immunogenicity. In general, the immunogenicity ofmouse antibodies is reduced by antibody humanization. It is assumed thatimmunogenicity risk can be further reduced by using a germline frameworksequence as a template in antibody humanization (Non-Patent document14). However, even Adalimumab, a fully human anti-TNF antibody, showedhigh-frequency (13% to 17%) immunogenicity, and the therapeutic effectwas found to be reduced in patients who showed immunogenicity(Non-Patent documents 15 and 16). T-cell epitopes may be present even inthe CDR of human antibodies, and these T-cell epitopes in CDR are apossible cause of immunogenicity. In silico and in vitro methods forpredicting T-cell epitopes have been reported (Non-Patent documents 17and 18). It is assumed that immunogenicity risk can be reduced byremoving T-cell epitopes predicted using such methods (Non-Patentdocument 19).

TOCILIZUMAB, a humanized anti-IL-6 receptor IgG1 antibody, is an IgG1antibody obtained by humanizing mouse antibody PM1. CDR grafting iscarried out using human NEW and REI sequences as template framework forH and L chains, respectively; however, five mouse sequence amino acidsare retained in the framework as essential amino acids for maintainingthe activity (Non-Patent Document 20). There is no previous report thatfully humanizes the remaining mouse sequence in the framework of thehumanized antibody TOCILIZUMAB without reducing the activity.Furthermore, the CDR sequence of TOCILIZUMAB is a mouse sequence, andthus, like Adalimumab, it may have T-cell epitopes in the CDR, which mayhave a potential immunogenicity risk. In clinical trials of TOCILIZUMAB,anti-TOCILIZUMAB antibodies were not detected at the effective dose of 8mg/kg, but they were observed at the doses of 2 mg/kg and 4 mg/kg(Patent Document 6). These suggest that there is still room forimprovement for the immunogenicity of TOCILIZUMAB. However, there hasbeen no report on reducing the immunogenicity risk of TOCILIZUMAB byamino acid substitution.

The isotype of TOCILIZUMAB is IgG1. The isotype difference refers todifference in the constant region sequence. Since the constant regionsequence is assumed to have strong influence on the effector function,pharmacokinetics, physical properties, and so on, selection of theconstant region sequence is very important for the development ofantibody pharmaceuticals (Non-Patent Document 11). In recent years, thesafety of antibody pharmaceuticals has become of great importance.Interaction between the antibody Fc portion and Fcγ receptor (effectorfunction) may have caused serious adverse effects in phase-I clinicaltrials of TGN1412 (Non-Patent Document 21). For antibody pharmaceuticalsdesigned to neutralize the biological activity of an antigen, thebinding to Fcγ receptor, which is important for effector functions suchas ADCC, is unnecessary. The binding to Fcγ receptor may even beunfavorable from the viewpoint of adverse effects. A method for reducingthe binding to Fcγ receptor is to alter the isotype of an IgG antibodyfrom IgG1 to IgG2 or IgG4 (Non-Patent Document 22). IgG2 is morefavorable than IgG4 from the viewpoint of pharmacokinetics and Fcγreceptor I binding (Non-Patent Document 11). TOCILIZUMAB is an IL-6receptor-neutralizing antibody, and its isotype is IgG1. Thus, in viewof the potential adverse effects, IgG2 may be a preferred isotype sinceeffector functions such as ADCC are not needed.

-   -   a. Meanwhile, when developing antibody pharmaceuticals,        physicochemical properties of the proteins, in particular,        homogeneity and stability are very crucial. It has been reported        that for the IgG2 isotype, there is significant heterogeneity        derived from the disulfide bonds in the hinge region (Non-Patent        Document 23). It is not easy and would be more costly to        manufacture them as pharmaceutical in large-scale while        maintaining the objective substances/related substances related        heterogeneity derived from disulfide bonds between productions.        Thus, single substances are desirable as much as possible.        Furthermore, for heterogeneity of the H-chain C-terminal        sequences of an antibody, deletion of C-terminal amino acid        lysine residue, and amidation of the C-terminal carboxyl group        due to deletion of both of the two C-terminal amino acids,        glycine and lysine, have been reported (Non-Patent Document 24).        In developing IgG2 isotype antibodies as pharmaceuticals, it is        preferable to reduce such heterogeneity and maintain high        stability. To produce convenient, stable, high-concentration,        subcutaneously-administered formulations, it is preferable that        not only the stability is high, but also the plasma half-life is        superior to that of IgG1 which is the isotype of TOCILIZUMAB.        However, there is no previous report on constant region        sequences for antibodies with the IgG2-isotype constant region        that have reduced heterogeneity, high stability, and superior        plasma half-life than antibodies with the IgG1 isotype constant        region.

Prior art documents related to the present invention are shown below:

[Prior Art Documents]

[Patent Documents]

-   [Patent Document 1] WO 92/19759-   [Patent Document 2] WO 96/11020-   [Patent Document 3] WO 96/12503-   [Patent Document 4] WO 2007/143168-   [Patent Document 5] WO 2007/114319-   [Patent Document 6] WO 2004/096273    [Non-Patent Documents]-   [Non-Patent Document 1] Janice M Reichert, Clark J Rosensweig, Laura    B Faden & Matthew C Dewitz, Monoclonal antibody successes in the    clinic, Nature Biotechnology 23, 1073-1078 (2005).-   [Non-Patent Document 2] Pavlou A K, Belsey M J., The therapeutic    antibodies market to 2008., Eur J Pharm Biopharm. 2005 April;    59(3):389-96.-   [Non-Patent Document 3] Nishimoto N, Kishimoto T., Interleukin 6:    from bench to bedside., Nat Clin Pract Rheumatol. 2006 November;    2(11):619-26.-   [Non-Patent Document 4] Maini R N, Taylor P C, Szechinski J, Pavelka    K, Broll J, Balint G, Emery P, Raemen F, Petersen J, Smolen J,    Thomson D, Kishimoto T; CHARISMA Study Group., Double-blind    randomized controlled clinical trial of the interleukin-6 receptor    antagonist, Tocilizumab, in European patients with rheumatoid    arthritis who had an incomplete response to methotrexate., Arthritis    Rheum. 2006 September; 54(9):2817-29.-   [Non-Patent Document 5] Nishimoto N, Kanakura Y, Aozasa K, Johkoh T,    Nakamura M, Nakano S, Nakano N, Ikeda Y, Sasaki T, Nishioka K, Hara    M, Taguchi H, Kimura Y, Kato Y, Asaoku H, Kumagai S, Kodama F,    Nakahara H, Hagihara K, Yoshizaki K, Kishimoto T. Humanized    anti-interleukin-6 receptor antibody treatment of multicentric    Castleman disease. Blood. 2005 Oct. 15; 106(8):2627-32.-   [Non-Patent Document 6] Kim S J, Park Y, Hong H J., Antibody    engineering for the development of therapeutic antibodies., Mol    Cells. 2005 Aug. 31; 20(1):17-29. Review.-   [Non-Patent Document 7] Rothe A, Hosse R J, Power B E. Ribosome    display for improved biotherapeutic molecules. Expert Opin Biol    Ther. 2006 February; 6(2):177-87.-   [Non-Patent Document 8] Rajpal A, Beyaz N, Haber L, Cappuccilli G,    Yee H, Bhatt R R, Takeuchi T, Lerner R A, Crea R., A general method    for greatly improving the affinity of antibodies by using    combinatorial libraries., Proc Natl Acad Sci USA. 2005 Jun. 14;    102(24):8466-71. Epub 2005 Jun. 6.-   [Non-Patent Document 9] Wu H, Pfarr D S, Johnson S, Brewah Y A,    Woods R M, Patel N K, White W I, Young J F, Kiener P A. Development    of Motavizumab, an Ultra-potent Antibody for the Prevention of    Respiratory Syncytial Virus Infection in the Upper and Lower    Respiratory Tract. J Mol Biol. 2007, 368, 652-665.-   [Non-Patent Document 10] Shire S J, Shahrokh Z, Liu J. Challenges in    the development of high protein concentration formulations. J Pharm    Sci. 2004 June; 93(6):1390-402.-   [Non-patent Document 11] Salfeld J G. Isotype selection in antibody    engineering. Nat Biotechnol. 2007 December; 25(12):1369-72.-   [Non-Patent Document 12] Hinton P R, Xiong J M, Johlfs M G, Tang M    T, Keller S, Tsurushita N., An engineered human IgG1 antibody with    longer serum half-life., J Immunol. 2006 Jan. 1; 176(1):346-56.-   [Non-Patent Document 13] Ghetie V, Popov S, Borvak J, Radu C,    Matesoi D, Medesan C, Ober R J, Ward E S., Increasing the serum    persistence of an IgG fragment by random mutagenesis., Nat    Biotechnol. 1997 July; 15(7):637-40.-   [Non-Patent Document 14] Hwang W Y, Almagro J C, Buss T N, Tan P,    Foote J. Use of human germline genes in a CDR homology-based    approach to antibody humanization. Methods. 2005 May; 36(1):35-42.-   [Non-Patent Document 15] Bartelds G M, Wijbrandts C A, Nurmohamed M    T, Stapel S, Lems W F, Aarden L, Dijkmans B A, Tak P, Wolbink G J.    Clinical response to adalimumab: The relationship with    anti-adalimumab antibodies and serum adalimumab concentrations in    rheumatoid arthritis Ann Rheum Dis. 2007 Mar. 9; [Epub ahead of    print]-   [Non-Patent Document 16] Bender N K, Heilig C E, Droll B, Wohlgemuth    J, Armbruster F P, Heilig B. Immunogenicity, efficacy and adverse    events of adalimumab in RA patients. Rheumatol Int. 2007 January;    27(3):269-74.-   [Non-Patent Document 17] Van Walle I, Gansemans Y, Parren P W, Stas    P, Lasters I. Immunogenicity screening in protein drug development.    Expert Opin Biol Ther. 2007 March; 7(3):405-18.-   [Non-Patent Document 18] Jones T D, Phillips W J, Smith B J, Bamford    C A, Nayee P D, Baglin T P, Gaston J S, Baker M P. Identification    and removal of a promiscuous CD4+ T cell epitope from the C1 domain    of factor VIII. J Thromb Haemost. 2005 May; 3(5):991-1000.-   [Non-Patent Document 19] Chirino A J, Ary M L, Marshall S A.    Minimizing the immunogenicity of protein therapeutics. Drug Discov    Today. 2004 Jan. 15; 9(2):82-90.-   [Non-Patent Document 20] Sato K, Tsuchiya M, Saldanha J, Koishihara    Y, Ohsugi Y, Kishimoto T, Bendig M M. Reshaping a human antibody to    inhibit the interleukin 6-dependent tumor cell growth. Cancer Res.    1993 Feb. 15; 53(4):851-6.-   [Non-Patent Document 21] Strand V, Kimberly R, Isaacs J D. Biologic    therapies in rheumatology: lessons learned future directions. Nat    Rev Drug Discov. 2007 January; 6(1):75-92.-   [Non-Patent Document 22] Gessner J E, Heiken H, Tamm A, Schmidt R E.    The IgG Fc receptor family. Ann Hematol. 1998 June; 76(6):231-48.-   [Non-Patent Document 23] Dillon T M, Ricci M S, Vezina C, Flynn G C,    Liu Y D, Rehder D S, Plant M, Henkle B, Li Y, Deechongkit S, Varnum    B, Wypych J, Balland A, Bondarenko P V. Structural and functional    characterization of disulfide isoforms of the human IgG2 subclass. J    Biol Chem. 2008 Jun. 6; 283(23):16206-15.-   [Non-Patent Document 24] Johnson K A, Paisley-Flango K, Tangarone B    S, Porter T J, Rouse J C. Cation exchange-HPLC and mass spectrometry    reveal C-terminal amidation of an IgG1 heavy chain. Anal Biochem.    2007 Jan. 1; 360(1):75-83.

DISCLOSURE OF THE INVENTION

[Problems to be Solved by the Invention]

The present invention was achieved in view of the above circumstances.An objective of the present invention is to provide pharmaceuticalcompositions that comprise second-generation molecules that are superiorthan the humanized anti-IL-6 receptor IgG1 antibody TOCILIZUMAB, byaltering the amino acid sequences of the variable and constant regionsof TOCILIZUMAB to enhance the antigen-neutralizing ability and improvepharmacokinetics, such that prolonged therapeutic effect is exerted witha less frequency of administration, and immunogenicity, safety, andphysicochemical properties (stability and homogeneity) are improved(hereinbelow, these pharmaceutical compositions may also be referred toas the “agents” or the “formulations”). Another objective is to providemethods for producing such pharmaceutical compositions.

[Means for Solving the Problems]

The present inventors conducted dedicated studies to generatesecond-generation molecules that are superior than the first-generationhumanized anti-IL-6 receptor IgG1 antibody TOCILIZUMAB, by altering theamino acid sequences of the variable and constant regions of TOCILIZUMABto enhance the efficacy and improve the pharmacokinetics, so thatprolonged therapeutic effect is exerted with a lower frequency ofadministration, and immunogenicity, safety, and physicochemicalproperties (stability and homogeneity) are improved. As a result, thepresent inventors discovered multiple CDR mutations in the variableregions of TOCILIZUMAB that improve the binding ability (affinity) tothe antigen. The present inventors thus successfully improved theaffinity significantly using a combination of such mutations. Thepresent inventors also succeeded in improving pharmacokinetics byintroducing modifications that lower the isoelectric point of thevariable region sequence. The present inventors also succeeded inimproving pharmacokinetics by making the binding to the IL-6 receptorantigen to be pH-dependent, so that a single antibody molecule canneutralize the antigen multiple times. Furthermore, the presentinventors successfully reduced the risk of immunogenicity by fullyhumanizing the mouse-derived sequences that remain in the framework ofTOCILIZUMAB and reducing the number of T-cell epitope peptides in thevariable regions predicted in silico. Furthermore, the present inventorsalso successfully discovered novel constant region sequences for theconstant region of TOCILIZUMAB, that reduce the binding to the Fcγreceptor as compared to IgG1 to improve safety, improve thepharmacokinetics as compared to IgG1, and reduce the heterogeneity dueto the disulfide bonds in the hinge region of IgG2 and the heterogeneitydue to the H chain C-terminus without decreasing stability. The presentinventors successfully produced second-generation molecules that aresuperior than TOCILIZUMAB by appropriately combining these amino acidsequence alterations in the CDR, variable regions, and constant regions.

The present invention relates to pharmaceutical compositions comprisinga humanized anti-IL-6 receptor IgG antibody having superior antigen(IL-6 receptor)-binding ability, superior pharmacokinetics, superiorsafety and physical properties (stability and homogeneity), and furtherreduced immunogenicity risk, by altering the amino acid sequences ofvariable and constant regions of the humanized anti-IL-6 receptor IgG1antibody TOCILIZUMAB; and methods for producing such pharmaceuticalcompositions. More specifically, the present invention provides:

-   [1] a polypeptide of any one of:    -   (a) a polypeptide that comprises CDR1 comprising the sequence of        SEQ ID NO: 1 (CDR1 of VH4-M73), CDR2 comprising the sequence of        SEQ ID NO: 2 (CDR2 of VH4-M73), and CDR3 comprising the sequence        of SEQ ID NO: 3 (CDR3 of VH4-M73);    -   (b) a polypeptide that comprises CDR1 comprising the sequence of        SEQ ID NO: 4 (CDR1 of VH3-M73), CDR2 comprising the sequence of        SEQ ID NO: 5 (CDR2 of VH3-M73), and CDR3 comprising the sequence        of SEQ ID NO: 6 (CDR3 of VH3-M73);    -   (c) a polypeptide that comprises CDR1 comprising the sequence of        SEQ ID NO: 7 (CDR1 of VH5-M83), CDR2 comprising the sequence of        SEQ ID NO: 8 (CDR2 of VH5-M83), and CDR3 comprising the sequence        of SEQ ID NO: 9 (CDR3 of VH5-M83);    -   (d) a polypeptide that comprises CDR1 comprising the sequence of        SEQ ID NO: 10 (CDR1 of VL1), CDR2 comprising the sequence of SEQ        ID NO: 11 (CDR2 of VL1), and CDR3 comprising the sequence of SEQ        ID NO: 12 (CDR3 of VL1);    -   (e) a polypeptide that comprises CDR1 comprising the sequence of        SEQ ID NO: 13 (CDR1 of VL3), CDR2 comprising the sequence of SEQ        ID NO: 14 (CDR2 of VL3), and CDR3 comprising the sequence of SEQ        ID NO: 15 (CDR3 of VL3); and    -   (f) a polypeptide that comprises CDR1 comprising the sequence of        SEQ ID NO: 16 (CDR1 of VL5), CDR2 comprising the sequence of SEQ        ID NO: 17 (CDR2 of VL5), and CDR3 comprising the sequence of SEQ        ID NO: 18 (CDR3 of VL5);-   [2] an antibody of any one of:    -   (a) an antibody which comprises a heavy chain variable region        that comprises CDR1 comprising the sequence of SEQ ID NO: 1        (CDR1 of VH4-M73), CDR2 comprising the sequence of SEQ ID NO: 2        (CDR2 of VH4-M73), and CDR3 comprising the sequence of SEQ ID        NO: 3 (CDR3 of VH4-M73), and a light chain variable region that        comprises CDR1 comprising the sequence of SEQ ID NO: 10 (CDR1 of        VL1), CDR2 comprising the sequence of SEQ ID NO: 11 (CDR2 of        VL1), and CDR3 comprising the sequence of SEQ ID NO: 12 (CDR3 of        VL1);    -   (b) an antibody which comprises a heavy chain variable region        that comprises CDR1 comprising the sequence of SEQ ID NO: 4        (CDR1 of VH3-M73), CDR2 comprising the sequence of SEQ ID NO: 5        (CDR2 of VH3-M73), and CDR3 comprising the sequence of SEQ ID        NO: 6 (CDR3 of VH3-M73), and a light chain variable region that        comprises CDR1 comprising the sequence of SEQ ID NO: 13 (CDR1 of        VL3), CDR2 comprising the sequence of SEQ ID NO: 14 (CDR2 of        VL3), and CDR3 comprising the sequence of SEQ ID NO: 15 (CDR3 of        VL3); and    -   (c) an antibody which comprises a heavy chain variable region        that comprises CDR1 comprising the sequence of SEQ ID NO: 7        (CDR1 of VH5-M83), CDR2 comprising the sequence of SEQ ID NO: 8        (CDR2 of VH5-M83), and CDR3 comprising the sequence of SEQ ID        NO: 9 (CDR3 of VH5-M83), and a light chain variable region that        comprises CDR1 comprising the sequence of SEQ ID NO: 16 (CDR1 of        VL5), CDR2 comprising the sequence of SEQ ID NO: 17 (CDR2 of        VL5), and CDR3 comprising the sequence of SEQ ID NO: 18 (CDR3 of        VL5);-   [3] a variable region of any one of:    -   (a) a heavy chain variable region comprising the sequence of SEQ        ID NO: 19 (variable region of VH4-M73);    -   (b) a heavy chain variable region comprising the sequence of SEQ        ID NO: 20 (variable region of VH3-M73);    -   (c) a heavy chain variable region comprising the sequence of SEQ        ID NO: 21 (variable region of VH5-M83);    -   (d) a light chain variable region comprising the sequence of SEQ        ID NO: 22 (variable region of VL1);    -   (e) a light chain variable region comprising the sequence of SEQ        ID NO: 23 (variable region of VL3); and    -   (f) a light chain variable region comprising the sequence of SEQ        ID NO: 24 (variable region of VL5);-   [4] an antibody of any one of:    -   (a) an antibody that comprises a heavy chain variable region        comprising the sequence of SEQ ID NO: 19 (variable region of        VH4-M73) and a light chain variable region comprising the        sequence of SEQ ID NO: 22 (variable region of VL1);    -   (b) an antibody that comprises a heavy chain variable region        comprising the sequence of SEQ ID NO: 20 (variable region of        VH3-M73) and a light chain variable region comprising the        sequence of SEQ ID NO: 23 (variable region of VL3); and    -   (c) an antibody that comprises a heavy chain variable region        comprising the sequence of SEQ ID NO: 21 (variable region of        VH5-M83) and a light chain variable region comprising the        sequence of SEQ ID NO: 24 (variable region of VL5);-   [5] a heavy chain or light chain of any one of:    -   (a) a heavy chain comprising the sequence of SEQ ID NO: 25        (VH4-M73);    -   (b) a heavy chain comprising the sequence of SEQ ID NO: 26        (VH3-M73);    -   (c) a heavy chain comprising the sequence of SEQ ID NO: 27        (VH5-M83);    -   (d) a light chain comprising the sequence of SEQ ID NO: 28        (VL1);    -   (e) a light chain comprising the sequence of SEQ ID NO: 29        (VL3); and    -   (f) a light chain comprising the sequence of SEQ ID NO: 30        (VL5);-   [6] an antibody of any one of:    -   (a) an antibody that comprises a heavy chain comprising the        sequence of SEQ ID NO: 25 (VH4-M73) and a light chain comprising        the sequence of SEQ ID NO: 28 (VL1);    -   (b) an antibody that comprises a heavy chain comprising the        sequence of SEQ ID NO: 26 (VH3-M73) and a light chain comprising        the sequence of SEQ ID NO: 29 (VL3); and    -   (c) an antibody that comprises a heavy chain comprising the        sequence of SEQ ID NO: 27 (VH5-M83) and a light chain comprising        the sequence of SEQ ID NO: 30 (VL5);-   [7] a gene encoding the polypeptide of any one of [1] to [6];-   [8] a vector carrying the gene of [7];-   [9] a host cell carrying the vector of [8];-   [10] a method for producing the polypeptide of any one of [1] to [6]    by culturing the host cell of [9]; and-   [11] a pharmaceutical composition comprising the polypeptide of any    one of [1] to [6] or a polypeptide produced by the method of [10].    [Effects of the Invention]

The humanized anti-IL-6 receptor IgG antibodies obtained according tothe present invention have enhanced efficacy and improvedpharmacokinetics; thus, they can exert a prolonged therapeutic effectwith a less administration frequency.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a listing of mutation sites that improve the affinity ofTOCILIZUMAB for the IL-6 receptor. The HCDR2 sequence of TOCILIZUMAB isshown in SEQ ID NO: 81; the HCDR2 sequence after mutation (upper line)is shown in SEQ ID NO: 82; the HCDR2 sequence after mutation (lowerline) is shown in SEQ ID NO: 83; the HCDR3 sequence of TOCILIZUMAB isshown in SEQ ID NO: 84; the HCDR3 sequence after mutation (upper line)is shown in SEQ ID NO: 85; the HCDR3 sequence after mutation (lowerline) is shown in SEQ ID NO: 86; the LCDR1 sequence of TOCILIZUMAB isshown in SEQ ID NO: 87; the LCDR1 sequence after mutation (upper line)is shown in SEQ ID NO: 88; the LCDR1 sequence after mutation (lowerline) is shown in SEQ ID NO: 89; the LCDR3 sequence of TOCILIZUMAB isshown in SEQ ID NO: 90; the LCDR3 sequence after mutation (upper line)is shown in SEQ ID NO: 91; and the LCDR3 sequence after mutation (lowerline) is shown in SEQ ID NO: 92.

FIG. 2 is a graph showing the neutralizing activities of TOCILIZUMAB andRDC-23 in BaF/gp 130.

FIG. 3 is a listing of mutation sites that can reduce the isoelectricpoint of variable region without significantly reducing the binding ofTOCILIZUMAB to the IL-6 receptor. Asterisk in the drawing represents asite that has no influence on the isoelectric point but which wasmutated for conversion into a human sequence. The HFR1 sequence ofTOCILIZUMAB is shown in SEQ ID NO: 93; the HFR1 sequence after mutationis shown in SEQ ID NO: 94; the HCDR1 sequence of TOCILIZUMAB is shown inSEQ ID NO: 95; the HCDR1 sequence after mutation is shown in SEQ ID NO:96; the HFR2 sequence of TOCILIZUMAB is shown in SEQ ID NO: 97; the HFR2sequence after mutation is shown in SEQ ID NO: 98; the HCDR2 sequence ofTOCILIZUMAB is shown in SEQ ID NO: 81; the HCDR2 sequence after mutationis shown in SEQ ID NO: 99; the HFR4 sequence of TOCILIZUMAB is shown inSEQ ID NO: 100; the HFR4 sequence after mutation is shown in SEQ ID NO:101; the LFR1 sequence of TOCILIZUMAB is shown in SEQ ID NO: 102; theLFR1 sequence after mutation is shown in SEQ ID NO: 103; the LCDR1sequence of TOCILIZUMAB is shown in SEQ ID NO: 87; the LCDR1 sequenceafter mutation is shown in SEQ ID NO: 104; the LFR2 sequence ofTOCILIZUMAB is shown in SEQ ID NO: 105; the LFR2 sequence after mutationis shown in SEQ ID NO: 106; the LCDR2 sequence of TOCILIZUMAB is shownin SEQ ID NO: 107; the LCDR2 sequences after mutation are shown in SEQID NOs: 108 and 109; the LFR3 sequence of TOCILIZUMAB is shown in SEQ IDNO: 110; the LFR3 sequence after mutation is shown in SEQ ID NO: 111;the LFR4 sequence of TOCILIZUMAB is shown in SEQ ID NO: 112; and theLFR4 sequence after mutation is shown in SEQ ID NO: 113.

FIG. 4 is a graph showing the neutralizing activities of TOCILIZUMAB andH53/L28 in BaF/gp130.

FIG. 5 is a graph showing the time courses of plasma concentration forTOCILIZUMAB and H53/L28 in mice after intravenous administration.

FIG. 6 is a graph showing the time courses of plasma concentration forTOCILIZUMAB and H53/L28 in mice after subcutaneous administration.

FIG. 7 is a schematic illustration showing that an IgG molecule can bindagain to another antigen by dissociating from a membrane-type antigen inthe endosome.

FIG. 8 is a listing of mutation sites that can confer pH dependency tothe binding of TOCILIZUMAB to the IL-6 receptor (binding at pH 7.4 anddissociation at pH 5.8). The HFR1 sequence of TOCILIZUMAB is shown inSEQ ID NO: 93; the HFR1 sequence after mutation is shown in SEQ ID NO:114; the HCDR1 sequence of TOCILIZUMAB is shown in SEQ ID NO: 95; theHCDR1 sequence after mutation is shown in SEQ ID NO: 115; the LCDR1sequence of TOCILIZUMAB is shown in SEQ ID NO: 87; the LCDR1 sequenceafter mutation is shown in SEQ ID NO: 116; the LCDR2 sequence ofTOCILIZUMAB is shown in SEQ ID NO: 107; and the LCDR2 sequence aftermutation is shown in SEQ ID NO: 117.

FIG. 9 is a graph showing the neutralizing activities of TOCILIZUMAB andH3pI/L73 in BaF/gp130.

FIG. 10 is a graph showing the time courses of plasma concentration forTOCILIZUMAB and H3pI/L73 in cynomolgus monkeys after intravenousadministration.

FIG. 11 is a graph showing the time courses of plasma concentration forTOCILIZUMAB and H3pI/L73 in human IL-6 receptor transgenic mice afterintravenous administration.

FIG. 12 is a diagram showing the result of assessment of theC-terminus-derived heterogeneity of TOCILIZUMAB, TOCILIZUMABAK, andTOCILIZUMABAGK by cation exchange chromatography.

FIG. 13 is a diagram showing the result of assessment of the disulfidebond-derived heterogeneity of TOCILIZUMAB-IgG1, TOCILIZUMAB-IgG2, andTOCILIZUMAB-SKSC by cation exchange chromatography.

FIG. 14 is a diagram showing the denaturation curves forTOCILIZUMAB-IgG1, TOCILIZUMAB-IgG2, and TOCILIZUMAB-SKSC obtained bydifferential scanning calorimetry (DSC), and the Tm value for each Fabdomain.

FIG. 15 is a graph showing the time courses of plasma concentration forTOCILIZUMAB-IgG1, TOCILIZUMAB-M44, TOCILIZUMAB-M58, and TOCILIZUMAB-M73in human FcRn transgenic mice after intravenous administration.

FIG. 16 is a graph showing the neutralizing activities of TOCILIZUMAB,control, and Fv5-M83 in BaF/gp130.

FIG. 17 is a graph showing the neutralizing activities of TOCILIZUMAB,Fv3-M73, and Fv4-M73 in BaF/gp130.

FIG. 18 is a graph showing the time courses of plasma concentrations forTOCILIZUMAB, control, Fv3-M73, Fv4-M73, and Fv5-M83 in cynomolgusmonkeys after intravenous administration.

FIG. 19 is a graph showing the time courses of CRP concentration forTOCILIZUMAB, control, Fv3-M73, Fv4-M73, or Fv5-M83 in cynomolgus monkeysafter intravenous administration.

FIG. 20 is a graph showing the time courses of percentage of freesoluble IL-6 receptor in cynomolgus monkeys after intravenousadministration of TOCILIZUMAB, control, Fv3-M73, Fv4-M73, or Fv5-M83.

FIG. 21 is a graph showing the inhibitory effects by TOCILIZUMAB andFv4-M73 on MCP-1 production from human RA patient-derived synovialcells.

FIG. 22 is a graph showing the inhibitory effects by TOCILIZUMAB andFv4-M73 on VEGF production from human RA patient-derived synovial cells.

MODE FOR CARRYING OUT THE INVENTION

The present invention provides the polypeptides of (a) to (f) below:

-   -   (a) a polypeptide that comprises CDR1 comprising the sequence of        SEQ ID NO: 1 (CDR1 of VH4-M73), CDR2 comprising the sequence of        SEQ ID NO: 2 (CDR2 of VH4-M73), and CDR3 comprising the sequence        of SEQ ID NO: 3 (CDR3 of VH4-M73);    -   (b) a polypeptide that comprises CDR1 comprising the sequence of        SEQ ID NO: 4 (CDR1 of VH3-M73), CDR2 comprising the sequence of        SEQ ID NO: 5 (CDR2 of VH3-M73), and CDR3 comprising the sequence        of SEQ ID NO: 6 (CDR3 of VH3-M73);    -   (c) a polypeptide that comprises CDR1 comprising the sequence of        SEQ ID NO: 7 (CDR1 of VH5-M83), CDR2 comprising the sequence of        SEQ ID NO: 8 (CDR2 of VH5-M83), and CDR3 comprising the sequence        of SEQ ID NO: 9 (CDR3 of VH5-M83);    -   (d) a polypeptide that comprises CDR1 comprising the sequence of        SEQ ID NO: 10 (CDR1 of VL1), CDR2 comprising the sequence of SEQ        ID NO: 11 (CDR2 of VL1), and CDR3 comprising the sequence of SEQ        ID NO: 12 (CDR3 of VL1);    -   (e) a polypeptide that comprises CDR1 comprising the sequence of        SEQ ID NO: 13 (CDR1 of VL3), CDR2 comprising the sequence of SEQ        ID NO: 14 (CDR2 of VL3), and CDR3 comprising the sequence of SEQ        ID NO: 15 (CDR3 of VL3); and    -   (f) a polypeptide that comprises CDR1 comprising the sequence of        SEQ ID NO: 16 (CDR1 of VL5), CDR2 comprising the sequence of SEQ        ID NO: 17 (CDR2 of VL5), and CDR3 comprising the sequence of SEQ        ID NO: 18 (CDR3 of VL5).

The polypeptides of the present invention are not particularly limited;however, they are preferably antigen-binding substances having theactivity of binding to human IL-6 receptor. Such antigen-bindingsubstances preferably include, for example, antibody heavy chainvariable regions (VH), antibody light chain variable regions (VL),antibody heavy chains, antibody light chains, and antibodies.

Of the polypeptides of (a) to (f) above, the polypeptides of (a) to (c)are preferable examples of antibody heavy chain variable regions, whilethe polypeptides of (d) to (f) are preferable examples of antibody lightchain variable regions.

These variable regions can be used as a portion of an anti-human IL-6receptor antibody. Anti-human IL-6 receptor antibodies in which such avariable region is used have superior binding activity, excellentpharmacokinetics, excellent safety, reduced immunogenicity, and/orsuperior physicochemical properties. In the present invention, excellentpharmacokinetics or improvement of pharmacokinetics refers to any oneof: decrease in “clearance (CL)”, increase in the “area under the curve(AUC)”, increase in “mean residence time”, and increase in “plasmahalf-life (t½)”, which are pharmacokinetic parameters calculated fromthe time course of plasma concentration when an antibody is administeredinto the body. Herein, superior physicochemical property or improvedphysicochemical property refers to, but is not limited to, improvedstability, decreased heterogeneity, or the like.

Human antibody framework regions (FRs) to be linked with CDR areselected so that the CDR forms a favorable antigen-binding site. FRs tobe used for the variable regions of the present invention are notparticularly limited and any FR may be used; however, human-derived FRsare preferably used. It is possible to use human-derived FRs having anatural sequence. Alternatively, if needed, substitution, deletion,addition and/or insertion or such of one or more amino acids may beintroduced into the framework region having a natural sequence so thatthe CDR forms an adequate antigen-binding site. Mutant FR sequenceshaving a desired property can be selected, for example, by measuring andevaluating the binding activity to an antigen for an antibody with an FRwith amino acid substitutions (Sato, K. et al., Cancer Res. (1993) 53,851-856).

Moreover, one or more amino acids may be substituted, deleted, added,and/or inserted in the CDR sequence described above. It is preferredthat a CDR sequence after substitution, deletion, addition, and/orinsertion of one or more amino acids has equivalent activity to the CDRsequence before alteration with regard to binding activity, neutralizingactivity, stability, immunogenicity, and/or pharmacokinetics. The numberof amino acids to be substituted, deleted, added, and/or inserted is notparticularly limited; however, it is preferably three amino acids orless, more preferably two amino acids or less, and still more preferablyone amino acid per CDR.

Methods for substituting one or more amino acid residues with otheramino acids of interest include, for example, site-directed mutagenesis(Hashimoto-Gotoh, T, Mizuno, T, Ogasahara, Y, and Nakagawa, M. (1995) Anoligodeoxyribonucleotide-directed dual amber method for site-directedmutagenesis. Gene 152, 271-275; Zoller, M J, and Smith, M. (1983)Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13vectors. Methods Enzymol. 100, 468-500; Kramer, W, Drutsa, V, Jansen, HW, Kramer, B, Pflugfelder, M, and Fritz, H J (1984) The gapped duplexDNA approach to oligonucleotide-directed mutation construction. NucleicAcids Res. 12, 9441-9456; Kramer W, and Fritz H J (1987)Oligonucleotide-directed construction of mutations via gapped duplex DNAMethods. Enzymol. 154, 350-367; Kunkel, T A (1985) Rapid and efficientsite-specific mutagenesis without phenotypic selection. Proc Natl AcadSci U.S.A. 82, 488-492). This method can be used to substitute desiredamino acids in an antibody with other amino acids of interest.Furthermore, amino acids in the frameworks and CDRs can be substitutedto other appropriate amino acids using library techniques such asframework shuffling (Mol. Immunol. 2007 April; 44(11): 3049-60) and CDRrepair (US 2006/0122377).

The present invention also provides the antibodies of (a) to (c) below:

-   -   (a) an antibody which comprises a heavy chain variable region        that comprises CDR1 comprising the sequence of SEQ ID NO: 1        (CDR1 of VH4-M73), CDR2 comprising the sequence of SEQ ID NO: 2        (CDR2 of VH4-M73), and CDR3 comprising the sequence of SEQ ID        NO: 3 (CDR3 of VH4-M73), and a light chain variable region that        comprises CDR1 comprising the sequence of SEQ ID NO: 10 (CDR1 of        VL1), CDR2 comprising the sequence of SEQ ID NO: 11 (CDR2 of        VL1), and CDR3 comprising the sequence of SEQ ID NO: 12 (CDR3 of        VL1);    -   (b) an antibody which comprises a heavy chain variable region        that comprises CDR1 comprising the sequence of SEQ ID NO: 4        (CDR1 of VH3-M73), CDR2 comprising the sequence of SEQ ID NO: 5        (CDR2 of VH3-M73), and CDR3 comprising the sequence of SEQ ID        NO: 6 (CDR3 of VH3-M73), and a light chain variable region that        comprises CDR1 comprising the sequence of SEQ ID NO: 13 (CDR1 of        VL3), CDR2 comprising the sequence of SEQ ID NO: 14 (CDR2 of        VL3), and CDR3 comprising the sequence of SEQ ID NO: 15 (CDR3 of        VL3); and    -   (c) an antibody which comprises a heavy chain variable region        that comprises CDR1 comprising the sequence of SEQ ID NO: 7        (CDR1 of VH5-M83), CDR2 comprising the sequence of SEQ ID NO: 8        (CDR2 of VH5-M83), and CDR3 comprising the sequence of SEQ ID        NO: 9 (CDR3 of VH5-M83), and a light chain variable region that        comprises CDR1 comprising the sequence of SEQ ID NO: 16 (CDR1 of        VL5), CDR2 comprising the sequence of SEQ ID NO: 17 (CDR2 of        VL5), and CDR3 comprising the sequence of SEQ ID NO: 18 (CDR3 of        VL5).

The antibodies described above can be used as anti-human IL-6 receptorantibodies having superior binding activity, excellent pharmacokinetics,excellent safety, reduced immunogenicity, and/or superiorphysicochemical properties.

Human antibody framework regions to be linked with CDR of the presentinvention are selected so that the CDR forms a favorable antigen-bindingsite. FRs to be used for the variable regions of the present inventionare not particularly limited, and any FR may be used; however,human-derived FR is preferably used. It is possible to use human-derivedFRs having a natural sequence. Alternatively, if needed, substitution,deletion, addition and/or insertion or such of one or more amino acidsmay be introduced into the framework region having a natural sequence sothat the CDR forms an adequate antigen-binding site. Mutant FR sequenceshaving a desired property can be selected, for example, by measuring andevaluating the binding activity to an antigen for an antibody having anFR with amino acid substitutions (Sato, K. et al., Cancer Res. (1993)53, 851-856).

Meanwhile, the constant region to be used for an antibody of the presentinvention is not particularly limited, and any constant region may beused. Preferred constant regions to be used for the antibodies of thepresent invention include, for example, human-derived constant regions(constant regions derived from IgG1, IgG2, IgG3, IgG4, Cκ, Cλ, andsuch). One or more amino acids may be substituted, deleted, added,and/or inserted in the human-derived constant regions. The preferredhuman-derived heavy chain constant regions include, for example,constant regions comprising the amino acid sequence of SEQ ID NO: 31(constant region of VH4-M73), constant regions comprising the amino acidsequence of SEQ ID NO: 32 (constant region VH3-M73)), and constantregions comprising the amino acid sequence of SEQ ID NO: 33 (constantregion of VH5-M83), while the preferred human-derived light chainconstant regions include, for example, constant regions comprising theamino acid sequence of SEQ ID NO: 34 (VL1), constant regions comprisingthe amino acid sequence of SEQ ID NO: 35 (VL3), and constant regionscomprising the amino acid sequence of SEQ ID NO: 36 (VL5).

Moreover, one or more amino acids may be substituted, deleted, added,and/or inserted in the CDR sequence described above. It is preferredthat a CDR sequence after substitution, deletion, addition, and/orinsertion of one or more amino acids has equivalent activity to the CDRsequence before alteration with regard to binding activity, neutralizingactivity, stability, immunogenicity, and/or pharmacokinetics. The numberof amino acids to be substituted, deleted, added, and/or inserted is notparticularly limited; however, it is preferably three amino acids orless, more preferably two amino acids or less, and still more preferablyone amino acid per CDR.

Amino acids can also be substituted, deleted, added, and/or inserted bythe methods described above.

The present invention also provides the variable regions of (a) to (f)below:

-   -   (a) a heavy chain variable region comprising the sequence of SEQ        ID NO: 19 (variable region of VH4-M73);    -   (b) a heavy chain variable region comprising the sequence of SEQ        ID NO: 20 (variable region of VH3-M73);    -   (c) a heavy chain variable region comprising the sequence of SEQ        ID NO: 21 (variable region of VH5-M83);    -   (d) a light chain variable region comprising the sequence of SEQ        ID NO: 22 (variable region of VL1);    -   (e) a light chain variable region comprising the sequence of SEQ        ID NO: 23 (variable region of VL3); and    -   (f) a light chain variable region comprising the sequence of SEQ        ID NO: 24 (variable region of VL5).

The variable regions described above can be used as part of ananti-human IL-6 receptor antibody. Anti-human IL-6 receptor antibodiesin which such variable regions are used have superior binding activity,excellent pharmacokinetics, excellent safety, reduced immunogenicity,and/or superior physicochemical properties.

The variable regions described above may also comprise substitutions,deletions, additions, and/or insertions of one or more amino acids (forexample, five amino acids or less, preferably three amino acids orless). Methods for substituting one or more amino acid residues withother amino acids of interest include, for example, the methodsdescribed above.

The present invention also provides polypeptides comprising the variableregions described above.

Furthermore, the present invention provides the antibodies of (a) to (c)below:

-   -   (a) an antibody that comprises a heavy chain variable region        comprising the sequence of SEQ ID NO: 19 (variable region of        VH4-M73) and a light chain variable region comprising the        sequence of SEQ ID NO: 22 (variable region of VL1);    -   (b) an antibody that comprises a heavy chain variable region        comprising the sequence of SEQ ID NO: 20 (variable region of        VH3-M73) and a light chain variable region comprising the        sequence of SEQ ID NO: 23 (variable region of VL3); and    -   (c) an antibody that comprises a heavy chain variable region        comprising the sequence of SEQ ID NO: 21 (variable region of        VH5-M83) and a light chain variable region comprising the        sequence of SEQ ID NO: 24 (variable region of VL5).

The variable regions described above can be used as part of ananti-human IL-6 receptor antibody. Anti-human IL-6 receptor antibodiesin which these variable regions are used have superior binding activity,excellent pharmacokinetics, excellent safety, reduced immunogenicity,and/or superior physical properties.

The variable regions described above may also comprise substitutions,deletions, additions, and/or insertions of one or more amino acids (forexample, five amino acids or less, preferably three amino acids orless). Methods for substituting one or more amino acid residues withother amino acids of interest include, for example, the methodsdescribed above.

Meanwhile, the constant region to be used for an antibody of the presentinvention is not particularly limited, and any constant region may beused. The preferred constant regions to be used for the antibodies ofthe present invention include, for example, human-derived constantregions (constant regions derived from IgG1, IgG2, IgG3, IgG4, κ chain,λ chain, and such). One or more amino acids may be substituted, deleted,added, and/or inserted in the human-derived constant regions. Thepreferred human-derived heavy chain constant regions include, forexample, constant regions comprising the amino acid sequence of SEQ IDNO: 31 (constant region of VH4-M73), constant regions comprising theamino acid sequence of SEQ ID NO: 32 (constant region VH3-M73)), andconstant regions comprising the amino acid sequence of SEQ ID NO: 33(constant region of VH5-M83), while the preferred human-derived lightchain constant regions include, for example, constant regions comprisingthe amino acid sequence of SEQ ID NO: 34 (VL1), constant regionscomprising the amino acid sequence of SEQ ID NO: 35 (VL3), and constantregions comprising the amino acid sequence of SEQ ID NO: 36 (VL5).

The present invention also provides the heavy or light chains of (a) to(f) below:

-   -   (a) a heavy chain comprising the sequence of SEQ ID NO: 25        (VH4-M73);    -   (b) a heavy chain comprising the sequence of SEQ ID NO: 26        (VH3-M73);    -   (c) a heavy chain comprising the sequence of SEQ ID NO: 27        (VH5-M83);    -   (d) a light chain comprising the sequence of SEQ ID NO: 28        (VL1);    -   (e) a light chain comprising the sequence of SEQ ID NO: 29        (VL3); and    -   (f) a light chain comprising the sequence of SEQ ID NO: 30        (VL5).

The heavy chains and light chains described above can be used as part ofan anti-human IL-6 receptor antibody. Anti-human IL-6 receptorantibodies in which these heavy chains and light chains are used havesuperior binding activity, excellent pharmacokinetics, excellent safety,reduced immunogenicity, and/or superior physicochemical properties.

The heavy chains and light chains described above may also comprisesubstitutions, deletions, additions, and/or insertions of one or moreamino acids (for example, ten amino acids or less, preferably five aminoacids or less, and more preferably three amino acids or less). Methodsfor substituting one or more amino acid residues with other amino acidsof interest include, for example, the methods described above.

Substitutions, deletions, additions, and/or insertions of one or moreamino acids may be carried out for the variable regions, constantregions, or both.

The present invention also provides the antibodies of (a) to (c) below:

-   -   (a) an antibody that comprises a heavy chain comprising the        sequence of SEQ ID NO: 25 (VH4-M73) and a light chain comprising        the sequence of SEQ ID NO: 28 (VL1);    -   (b) an antibody that comprises a heavy chain comprising the        sequence of SEQ ID NO: 26 (VH3-M73) and a light chain comprising        the sequence of SEQ ID NO: 29 (VL3); and    -   (c) an antibody that comprises a heavy chain comprising the        sequence of SEQ ID NO: 27 (VH5-M83) and a light chain comprising        the sequence of SEQ ID NO: 30 (VL5).

The antibodies described above are anti-human IL-6 receptor antibodiesthat have superior binding activity, excellent pharmacokinetics,excellent safety, reduced immunogenicity, and/or superiorphysicochemical properties.

The antibodies described above may also comprise substitutions,deletions, additions, and/or insertions of one or more amino acids (forexample, 20 amino acids or less, preferably ten amino acids or less, andmore preferably five amino acids or less). Methods for substituting oneor more amino acid residues with other amino acids of interest include,for example, the methods described above.

Substitutions, deletions, additions, and/or insertions of one or moreamino acids may be carried out for the variable regions, constantregions, or both.

The antibodies of the present invention are preferably humanizedantibodies.

Humanized antibodies are also referred to as reshaped human antibodies.Such a humanized antibody is obtained by grafting a complementarydetermining region (CDR) derived from a non-human mammal into the CDR ofa human antibody. Conventional genetic recombination techniques for thepreparation of such antibodies are also known (see European PatentApplication No. EP 125023; and WO 96/02576).

Specifically, for example, a DNA sequence designed such that a CDR ofinterest and a framework region (FR) of interest are linked issynthesized by PCR, using several oligonucleotides prepared to haveoverlapping portions with the ends of both CDR and FR as primers (seethe method described in WO 98/13388). A humanized antibody is obtainedby: ligating the resulting DNA to a DNA that encodes a human antibodyconstant region or a modified human antibody constant region; insertingthis into an expression vector; and introducing this into a host toproduce the antibody (see European Patent Application No. EP 239400 andInternational Patent Application Publication No. WO 96/02576).

Human antibody framework regions to be linked with CDR are selected sothat the CDR forms a favorable antigen-binding site. If needed, aminoacid substitution, deletion, addition and/or insertion may be introducedinto the framework region of an antibody variable region.

A human antibody constant region, or an altered human antibody constantregion in which one or more amino acids have been substituted, deleted,added, and/or inserted in a human antibody constant region, can be usedas the constant region of a humanized antibody.

For example, Cγ1, Cγ2, Cγ3, Cγ4, Cμ, Cδ, Cα1, Cα2, and Cε can be usedfor the H chain, and Cκ and Cλ can be used for the L chain. The aminoacid sequence of Cκ is shown in SEQ ID NO: 38, and the nucleotidesequence encoding this amino acid sequence is shown in SEQ ID NO: 37.The amino acid sequence of Cγ1 is shown in SEQ ID NO: 40, and thenucleotide sequence encoding this amino acid sequence is shown in SEQ IDNO: 39. The amino acid sequence of Cγ2 is shown in SEQ ID NO: 42, andthe nucleotide sequence encoding this amino acid sequence is shown inSEQ ID NO: 41. The amino acid sequence of Cγ4 is shown in SEQ ID NO: 44,and the nucleotide sequence encoding this amino acid sequence is shownin SEQ ID NO: 43.

Furthermore, human antibody C regions may be modified to improveantibody stability or antibody production stability. Human antibodies ofany isotype such as IgG, IgM, IgA, IgE, or IgD may be used in antibodyhumanization; however, IgG is preferably used in the present invention.IgG1, IgG2, IgG3, IgG4, or the like can be used as the IgG.

Amino acids in the variable region (for example, CDR and FR) andconstant region of a humanized antibody may be deleted, added, inserted,and/or substituted with amino acids after preparation. The antibodies ofthe present invention also include such humanized antibodies comprisingamino acid substitutions and the like.

The antibodies of the present invention include not only divalentantibodies as represented by IgG, but also monovalent antibodies andmultivalent antibodies as represented by IgM, as long as they have IL-6receptor-binding activity and/or neutralizing activity. The multivalentantibodies of the present invention include multivalent antibodies inwhich the antigen-binding sites are all identical, and multivalentantibodies in which all or some of the antigen-binding sites aredifferent. The antibodies of the present invention include not onlywhole antibody molecules, but also minibodies and modified productsthereof, as long as they bind to the IL-6 receptor protein.

Minibodies are antibodies comprising an antibody fragment lacking aportion of a whole antibody (for example, whole IgG or such), and arenot particularly limited as long as they have IL-6 receptor-bindingactivity and/or neutralizing activity and comprise an antibody fragmentthat lacks a portion of a whole antibody (for example, whole IgG orsuch). The minibodies of the present invention are not particularlylimited, as long as they comprise a portion of a whole antibody.However, the minibodies preferably comprise VH or VL, and particularlypreferably comprise both VH and VL. Other preferable minibodies of thepresent invention include, for example, minibodies comprising antibodyCDRs. The minibodies may comprise all or some of the six CDRs of anantibody.

The minibodies of the present invention preferably have a smallermolecular weight than whole antibodies. However, the minibodies may formmultimers, for example, dimers, trimers, or tetramers, and thus theirmolecular weight is sometimes greater than that of whole antibodies.

Specifically, antibody fragments include, for example, Fab, Fab′,F(ab′)2, and Fv. Meanwhile, minibodies include, for example, Fab, Fab′,F(ab′)2, Fv, scFv (single chain Fv), diabodies, and sc(Fv)2 (singlechain (Fv)2). Multimers (for example, dimers, trimers, tetramers, andpolymers) of these antibodies are also included in the minibodies of thepresent invention.

Antibody fragments can be obtained, for example, by treating antibodieswith enzymes to produce antibody fragments. Enzymes known to generateantibody fragments include, for example, papain, pepsin, and plasmin.Alternatively, a gene encoding such antibody fragment can beconstructed, introduced into an expression vector, and expressed inappropriate host cells (see, for example, Co, M. S. et al., J. Immunol.(1994) 152, 2968-2976; Better, M. & Horwitz, A. H. Methods in Enzymology(1989) 178, 476-496; Pluckthun, A. & Skerra, A. Methods in Enzymology(1989) 178, 476-496; Lamoyi, E., Methods in Enzymology (1989) 121,652-663; Rousseaux, J. et al., Methods in Enzymology (1989) 121,663-669; Bird, R. E. et al., TIBTECH (1991) 9, 132-137).

Digestive enzymes cleave at specific sites of an antibody fragment,yielding antibody fragments of specific structures shown below. Geneticengineering techniques can be applied to such enzymatically-obtainedantibody fragments to delete an arbitrary portion of the antibody.

Antibody fragments obtained by using the above digestive enzymes are asfollows.

-   Papain digestion: F(ab)2 or Fab-   Pepsin digestion: F(ab′)2 or Fab′-   Plasmin digestion: Facb

The minibodies of the present invention include antibody fragmentslacking an arbitrary region, as long as they have IL-6 receptor-bindingactivity and/or neutralizing activity.

“Diabody” refers to a bivalent antibody fragment constructed by genefusion (Holliger P et al., 1993, Proc. Natl. Acad. Sci. USA 90:6444-6448; EP 404,097; WO 93/11161, etc). Diabodies are dimers composedof two polypeptide chains. In each of the polypeptide chains forming adimer, a VL and a VH are generally linked by a linker in the same chain.In general, a linker in a diabody is short enough such that the VL andVH cannot bind to each other. Specifically, the number of amino acidresidues constituting the linker is, for example, about five residues.Thus, the VL and VH encoded on the same polypeptide cannot form asingle-chain variable region fragment, and will form a dimer withanother single-chain variable region fragment. As a result, the diabodyhas two antigen binding sites.

ScFv antibodies are single-chain polypeptides produced by linking VH andVL via a linker or such (Huston, J. S. et al., Proc. Natl. Acad. Sci.U.S.A. (1988) 85, 5879-5883; Pluckthun “The Pharmacology of MonoclonalAntibodies” Vol. 113, eds., Resenburg and Moore, Springer Verlag, NewYork, pp. 269-315, (1994)). The H-chain V region and L-chain V region ofscFv may be derived from any antibody described herein. The peptidelinker for linking the V regions is not particularly limited. Forexample, an arbitrary single-chain peptide containing about three to 25residues can be used as the linker. Specifically, it is possible to usethe peptide linkers described below or such.

The V regions of the two chains can be linked, for example, by PCR asdescribed above. First, a DNA encoding the complete amino acid sequenceor a desired partial amino acid sequence of one of the DNAs shown belowis used as a template to link the V regions by PCR:

-   a DNA sequence encoding an H chain or H-chain V region of an    antibody, and-   a DNA sequence encoding an L chain or L-chain V region of an    antibody.

DNAs encoding the V region of an H chain or L chain are amplified by PCRusing a pair of primers containing corresponding sequences of the twoends of the DNA to be amplified. Then, a DNA encoding the peptide linkerportion is prepared. The peptide linker-encoding DNA can also besynthesized by PCR. A nucleotide sequence that can be used to link theseparately synthesized amplification products of V region is added tothe 5′ end of the primers to be used. Then, PCR is carried out usingeach of the DNAs in [H chain V region DNA]-[peptide linker DNA]-[L chainV region DNA] and assembly PCR primers.

The assembly PCR primers contain a combination of a primer that annealswith the 5′ end of the [H chain V region DNA] and a primer that annealswith the 3′ end of the [L chain V region DNA]. In other words, theassembly PCR primers are a set of primers that can be used to amplifyDNAs encoding the full-length sequence of the scFv to be synthesized.Meanwhile, nucleic sequences that can be used to link each of theV-region DNAs are added to the [peptide linker DNA]. Then, these DNAsare linked, and then the whole scFv is ultimately generated as anamplification product using the assembly PCR primers. Once thescFv-encoding DNAs are generated, expression vectors containing theseDNAs and recombinant cells transformed with these expression vectors canbe obtained by conventional methods. Further, the scFv can be obtainedthrough expression of the scFv-encoding DNAs by culturing the resultingrecombinant cells.

The order of VH and VL to be linked is not particularly limited, andthey may be arranged in any order. Examples of the arrangement arelisted below.

-   [VH] linker [VL]-   [VL] linker [VH]

sc(Fv)2 is a single-chain minibody produced by linking two VHs and twoVLs using linkers and such (Hudson et al., 1999, J Immunol. Methods231:177-189). sc(Fv)2 can be produced, for example, by linking scFvusing a linker.

Preferably, the two VHs and two VLs of an antibody are arranged in theorder of VH, VL, VH, and VL ([VH] linker [VL] linker [VH] linker [VL])from the N terminus of the single-chain polypeptide; however, the orderof the two VHs and two VLs is not limited to the above arrangement, andthey may be arranged in any order. Examples of the arrangement arelisted below:

-   [VL] linker [VH] linker [VH] linker [VL]-   [VH] linker [VL] linker [VL] linker [VH]-   [VH] linker [VH] linker [VL] linker [VL]-   [VL] linker [VL] linker [VH] linker [VH]-   [VL] linker [VH] linker [VL] linker [VH]

The amino acid sequence of the minibody VH or VL may containsubstitutions, deletions, additions, and/or insertions. Furthermore, aslong as VH and VL have antigen-binding activity when assembled, aportion may be deleted or other polypeptides may be added. Moreover, thevariable regions may be chimerized or humanized.

In the present invention, linkers that can be used to link the antibodyvariable regions include arbitrary peptide linkers that can beintroduced by genetic engineering, and synthetic linkers, for example,the linkers disclosed in Protein Engineering, (1996) 9(3), 299-305.

The preferred linkers in the present invention are peptide linkers. Thelength of the peptide linkers is not particularly limited and thoseskilled in the art can appropriately select the length according to thepurpose. The typical length is one to 100 amino acids, preferably 3 to50 amino acids, more preferably 5 to 30 amino acids, and particularlypreferably 12 to 18 amino acids (for example, 15 amino acids).

For example, amino acid sequences for peptide linkers include thefollowing sequences:

Ser Gly•Ser Gly•Gly•Ser Ser•Gly•Gly Gly•Gly•Gly•Ser  (SEQ ID NO: 45)Ser•Gly•Gly•Gly  (SEQ ID NO: 46) Gly•Gly•Gly•Gly•Ser  (SEQ ID NO: 47)Ser•Gly•Gly•Gly•Gly  (SEQ ID NO: 48) Gly•Gly•Gly•Gly•Gly•Ser (SEQ ID NO: 49) Ser•Gly•Gly•Gly•Gly•Gly  (SEQ ID NO: 50)Gly•Gly•Gly•Gly•Gly•Gly•Ser  (SEQ ID NO: 51)Ser•Gly•Gly•Gly•Gly•Gly•Gly  (SEQ ID NO: 52) (Gly•Gly•Gly•Gly•Ser)n[SEQ ID NO: 47] (Ser•Gly•Gly•Gly•Gly)n [SEQ ID NO: 48]where n is an integer of 1 or more.

The amino acid sequences of peptide linkers can be appropriatelyselected by those skilled in the art according to the purpose. Forexample, the above “n” which determines the length of the peptide linkeris typically one to five, preferably one to three, and more preferablyone or two.

Synthetic linkers (chemical crosslinking agents) include, crosslinkingagents routinely used to crosslink peptides, for example,N-hydroxysuccinimide (NHS), disuccinimidyl suberate (DSS),bis(sulfosuccinimidyl)suberate (BS3), dithiobis(succinimidyl propionate)(DSP), dithiobis(sulfosuccinimidyl propionate) (DTSSP), ethylene glycolbis(succinimidyl succinate) (EGS), ethylene glycol bis(sulfosuccinimidylsuccinate) (sulfo-EGS), disuccinimidyl tartarate (DST),disulfosuccinimidyl tartarate (sulfo-DST),bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone (BSOCOES), andbis[2-(sulfosuccinimidooxycarbonyloxy)ethyl]sulfone (sulfo-BSOCOES).These crosslinking agents are commercially available.

In general, three linkers are required to link four antibody variableregions. These multiple linkers may be the same or different linkers.

The antibodies of the present invention also include antibodies in whichone or more amino acid residues have been added to the amino acidsequence of an antibody of the present invention. Furthermore, theantibodies of the present invention also include fusion proteins inwhich an above-described antibody is fused with another peptide orprotein. The fusion protein can be prepared by ligating a polynucleotideencoding an antibody of the present invention and a polynucleotideencoding another peptide or polypeptide in frame, introducing this intoan expression vector, and expressing this in a host. Techniques known tothose skilled in the art can be used. The peptide or polypeptide to befused with an antibody of the present invention may be a known peptide,for example, FLAG (Hopp, T. P. et al., BioTechnology 6, 1204-1210(1988)), 6× His consisting of six His (histidine) residues, 10×His,influenza hemagglutinin (HA), human c-myc fragment, VSV-GP fragment,p18HIV fragment, T7-tag, HSV-tag, E-tag, SV40 T antigen fragment, lcktag, α-tubulin fragment, B-tag, and Protein C fragment. Polypeptides tobe fused with the antibodies of the present invention include, forexample, GST (glutathione-S-transferase), HA (influenza hemagglutinin),immunoglobulin constant region, β-galactosidase, and MBP(maltose-binding protein). Commercially available polynucleotidesencoding these peptides or polypeptides can be fused with apolynucleotide encoding an antibody of the present invention. A fusionpolypeptide can be prepared by expressing the fusion polynucleotide thusprepared.

Moreover, the antibodies of the present invention may also be conjugatedantibodies linked to various molecules such as polymers, includingpolyethylene glycol (PEG) and hyaluronic acid; radioactive substances;fluorescent substances; luminescent substances; enzymes; and toxins.Such conjugated antibodies can be obtained by chemically modifying theobtained antibodies. Methods for antibody modification are alreadyestablished in the art (see, for example, U.S. Pat. No. 5,057,313 andU.S. Pat. No. 5,156,840). The “antibodies” of the present invention alsoinclude such conjugated antibodies.

Furthermore, the antibodies of the present invention include antibodieswith altered sugar chains.

Furthermore, the antibodies used in the present invention may bebispecific antibodies. Bispecific antibody refers to an antibody thathas variable regions that recognize different epitopes in the sameantibody molecule. A bispecific antibody of the present invention may bea bispecific antibody that recognizes different epitopes on the IL-6receptor molecule, or a bispecific antibody in which one of theantigen-binding sites recognizes the IL-6 receptor and the otherantigen-binding site recognizes another substance. Examples of antigensthat bind to the other antigen-binding site of a bispecific antibodythat comprises an IL-6 receptor-recognizing antibody of the presentinvention include IL-6, TNFα, TNFR1, TNFR2, CD80, CD86, CD28, CD20,CD19, IL-1α, IL-β, IL-1R, RANKL, RANK, IL-17, IL-17R, IL-23, IL-23R,IL-15, IL-15R, BlyS, lymphotoxin α, lymphotoxin β, LIGHT ligand, LIGHT,VLA-4, CD25, IL-12, IL-12R, CD40, CD40L, BAFF, CD52, CD22, IL-32, IL-21,IL-21R, GM-CSF, GM-CSFR, M-CSF, M-CSFR, IFN-alpha, VEGF, VEGFR, EGF,EGFR, CCR5, APRIL, and APRILR.

Methods for producing bispecific antibodies are known. Bispecificantibodies can be prepared, for example, by linking two types ofantibodies recognizing different antigens. Antibodies to be linked maybe a half molecule each containing an H chain and an L chain, or aquarter molecule containing only one H chain. Alternatively, fusioncells producing bispecific antibodies can be prepared by fusinghybridomas producing different monoclonal antibodies. Furthermore,bispecific antibodies can be produced by genetic engineering techniques.

As described below, the antibodies of the present invention may differin amino acid sequence, molecular weight, isoelectric point,presence/absence of sugar chains, and conformation, depending on thepurification method, or the cell or host used to produce the antibodies.However, as long as the antibody obtained is functionally equivalent toan antibody of the present invention, it is included in the presentinvention. For example, when an antibody of the present invention isexpressed in prokaryotic cells, for example, Escherichia coli, amethionine residue is added to the N terminus of the original antibodyamino acid sequence. Such antibodies are also included in the antibodiesof the present invention.

Polypeptides of anti-IL-6 receptor antibodies and such of the presentinvention can be produced by methods known to those skilled in the art.

An anti-IL-6 receptor antibody can be prepared, for example, by geneticrecombination techniques known to those skilled in the art based on thesequence of the anti-IL-6 receptor antibody obtained. Specifically, ananti-IL-6 receptor antibody can be prepared by constructing apolynucleotide encoding the antibody based on the sequence of an IL-6receptor-recognizing antibody, inserting the polynucleotide into anexpression vector, and then expressing it in an appropriate host cell(see for example, Co, M. S. et al., J. Immunol. (1994) 152, 2968-2976;Better, M. and Horwitz, A. H., Methods Enzymol. (1989) 178, 476-496;Pluckthun, A. and Skerra, A., Methods Enzymol. (1989) 178, 497-515;Lamoyi, E., Methods Enzymol. (1986) 121, 652-663; Rousseaux, J. et al.,Methods Enzymol. (1986) 121, 663-669; Bird, R. E. and Walker, B. W.,Trends Biotechnol. (1991) 9, 132-137).

Thus, the present invention provides methods of producing (i) apolypeptide of the present invention, or (ii) a polypeptide encoded by agene encoding the polypeptide of the present invention, wherein themethods comprise the step of culturing a host cell comprising a vectorinto which a polynucleotide encoding the polypeptide of the presentinvention is introduced.

More specifically, the present invention provides methods of producing apolypeptide of the present invention, which comprise the steps of:

(a) culturing a host cell comprising a vector into which a gene encodingthe polypeptide of the present invention is introduced; and

(b) obtaining the polypeptide encoded by the gene.

Examples of the vector include M13-type vectors, pUC-type vectors,pBR322, pBluescript, and pCR-Script. Alternatively, when the objectiveis to subclone and excise the cDNA, other examples of the vector inaddition to the ones described above include pGEM-T, pDIRECT, and pT7.Expression vectors are particularly useful for producing antibodies ofthe present invention. For example, when the expression vector is usedfor expression in E. coli, the vector should have features that allowits amplification in E. coli. In addition, when the host is E. coli suchas JM109, DH5α, HB101, or XL1-Blue, it is essential that the vectorcarries a promoter that allows its efficient expression in E. coli, forexample, lacZ promoter (Ward et al., Nature (1989) 341, 544-546; FASEBJ. (1992) 6, 2422-2427), araB promoter (Better et al., Science (1988)240, 1041-1043), T7 promoter or such. Such vector includes pGEX-5X-1(Pharmacia), “QIAexpress system” (Quiagen), pEGFP, and pET (in thiscase, the host is preferably BL21 which expresses T7 RNA polymerase), inaddition to the ones described above.

Furthermore, the expression plasmid vectors may contain signal sequencesfor antibody secretion. As a signal sequence for antibody secretion, thepelB signal sequence (Lei, S. P. et al., J. Bacteriol. (1987) 169, 4379)may be used for production into the E. coli periplasm. The vectors canbe introduced into host cells, for example, by calcium chloride methodsor electroporation.

In addition to vectors for E. coli, the vectors for producing antibodiesof the present invention include, for example, mammal-derived expressionvectors (for example, pcDNA3 (Invitrogen), pEF-BOS (Nucleic Acids. Res.(1990) 18(17), p5322), pEF, and pCDM8), insect cell-derived expressionvectors (for example, the “Bac-to-BAC baculovirus expression system”(Gibco-BRL) and pBacPAK8), plant-derived expression vectors (forexample, pMH1 and pMH2), animal virus-derived expression vectors (forexample, pHSV, pMV, and pAdexLcw), retrovirus-derived expression vectors(for example, pZlPneo), yeast-derived expression vectors (for example,“Pichia Expression Kit” (Invitrogen), pNV11, and SP-Q01), and Bacillussubtilis-derived expression vectors (for example, pPL608 and pKTH50).

When the expression plasmid vector is used for expression in animalcells such as CHO, COS, and NIH3T3 cells, it must have a promoternecessary for expression in those cells, for example, SV40 promoter(Mulligan et al., Nature (1979) 277, 108), MMLV-LTR promoter, EF1αpromoter (Mizushima et al., Nucleic Acids Res. (1990) 18, 5322), or CMVpromoter. It is even more preferable if the vector has a gene forselection of transformed cells (for example, a drug resistance gene thatallows distinction by an agent (neomycin, G418, or such). Vectors withsuch characteristics include, for example, pMAM, pDR2, pBK-RSV, pBK-CMV,pOPRSV, and pOP13.

In addition, when the objective is to stably express genes and amplify agene's copy number in the cells, a method in which CHO cells deficientin a nucleic acid synthesis pathway are introduced with a vector havinga DHFR gene which compensates for the deficiency (for example, pSV2-dhfr(“Molecular Cloning 2nd edition” Cold Spring Harbor Laboratory Press,(1989))) and the vector is amplified using methotrexate (MTX) can beused. Further, when the objective is transient gene expression, a methodin which COS cells carrying a gene expressing the SV40 T antigen ontheir chromosome are transformed with a vector carrying an SV40replication origin (pcD and such) can be used. It is possible to usereplication origins derived from polyoma virus, adenovirus, bovinepapilloma virus (BPV), and such. Moreover, to amplify the gene copynumber in host cell lines, the expression vectors may comprise theaminoglycoside transferase (APH) gene, thymidine kinase (TK) gene, E.coli xanthine-guanine phosphoribosyltransferase (Ecogpt) gene,dihydrofolate reductase (dhfr) gene, and such as a selection marker.

The resulting antibodies of the present invention can be isolated fromhost cells or from outside the cells (the medium, or such), and purifiedas substantially pure and homogenous antibodies. The antibodies can beseparated and purified using conventional separation and purificationmethods for antibody purification, without being limited thereto. Forexample, the antibodies can be separated and purified by appropriatelyselecting and combining column chromatography, filtration,ultrafiltration, salting out, solvent precipitation, solvent extraction,distillation, immunoprecipitation, SDS-polyacrylamide gelelectrophoresis, isoelectrofocusing, dialysis, recrystallization, andsuch.

Chromatography includes, for example, affinity chromatography, ionexchange chromatography, hydrophobic chromatography, gel filtration,reverse phase chromatography, and adsorption chromatography (Strategiesfor Protein Purification and Characterization: A Laboratory CourseManual. Ed Daniel R. Marshak et al., Cold Spring Harbor LaboratoryPress, 1996). These chromatographies can be carried out usingliquid-phase chromatography, for example, HPLC and FPLC. Columns usedfor affinity chromatography include protein A columns and protein Gcolumns. Examples of columns using Protein A include Hyper D, POROS, andSepharose FF (GE Amersham Biosciences). The present invention alsoincludes antibodies highly purified using such purification methods.

The IL-6 receptor binding activity of the obtained antibodies can bemeasured by methods known to those skilled in the art. Methods formeasuring the antigen-binding activity of an antibody include, forexample, enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay(EIA), radioimmunoassay (RIA), and fluorescent antibody methods. Forexample, when enzyme immunoassay is used, antibody-containing samplessuch as purified antibodies and culture supernatants ofantibody-producing cells are added to antigen-coated plates. A secondaryantibody labeled with an enzyme such as alkaline phosphatase is added,and the plates are incubated. After washing, an enzyme substrate such asp-nitrophenyl phosphate is added, and the absorbance is measured toevaluate the antigen-binding activity.

Pharmaceutical Compositions

The present invention also provides pharmaceutical compositions thatcomprise an above-described polypeptide as an active ingredient. Thepharmaceutical compositions of the present invention can be used forIL-6-associated diseases such as rheumatoid arthritis. Thus, the presentinvention also provides agents for treating diseases such as rheumatoidarthritis, which comprise an antibody described above as an activeingredient. Preferred examples of target diseases in the presentinvention include, but are not limited to, rheumatoid arthritis,juvenile idiopathic arthritis, systemic juvenile idiopathic arthritis,Castleman's disease, systemic lupus erythematosus (SLE), lupusnephritis, Crohn's disease, lymphoma, ulcerative colitis, anemia,vasculitis, Kawasaki disease, Still's disease, amyloidosis, multiplesclerosis, transplantation, age-related macular degeneration, ankylosingspondylitis, psoriasis, psoriatic arthritis, chronic obstructivepulmonary disease (COPD), IgA nephropathy, osteoarthritis, asthma,diabetic nephropathy, GVHD, endometriosis, hepatitis (NASH), myocardialinfarction, arteriosclerosis, sepsis, osteoporosis, diabetes, multiplemyeloma, prostate cancer, kidney cancer, B-cell non-Hodgkin's lymphoma,pancreatic cancer, lung cancer, esophageal cancer, colon cancer, cancercachexia, cancer neuroinvasion, myocardial infarction, myopic choroidalneovascularization, idiopathic choroidal neovascularization, uveitis,chronic thyroiditis, delayed hypersensitivity, contact dermatitis,atopic dermatitis, mesothelioma, polymyositis, dermatomyositis,panuveitis, anterior uveitis, intermediate uveitis, scleritis,keratitis, orbital inflammation, optic neuritis, diabetic retinopathy,proliferative vitreoretinopathy, dry eye, and post-operativeinflammation.

The phrase “to comprise an anti-IL-6 receptor antibody as an activeingredient” means comprising an anti-IL-6 receptor antibody as at leastone of the active ingredients, without particular limitation on itscontent. Furthermore, the pharmaceutical compositions of the presentinvention may contain other active ingredients in combination with thepolypeptides described above.

The pharmaceutical compositions of the present invention may be used notonly for therapeutic purposes, but also for preventive purposes.

The polypeptides of the present invention can be formulated according toconventional methods (see, for example, Remington's PharmaceuticalScience, latest edition, Mark Publishing Company, Easton, USA). Ifneeded, they may contain pharmaceutically acceptable carriers and/oradditives. For example, they may include detergents (for example, PEGand Tween), excipients, antioxidants (for example, ascorbic acid),coloring agents, flavoring agents, preservatives, stabilizers, bufferingagents (for example, phosphoric acid, citric acid, and other organicacids), chelating agents (for example, EDTA), suspending agents,isotonizing agents, binders, disintegrants, lubricants, fluiditypromoters, and corrigents. However, the agents of the present inventionfor preventing or treating inflammatory diseases are not limited to theabove and may appropriately contain other conventional carriers.Specifically, examples include light anhydrous silicic acid, lactose,crystalline cellulose, mannitol, starch, carmellose calcium, carmellosesodium, hydroxypropylcellulose, hydroxypropyl methylcellulose, polyvinylacetal diethylaminoacetate, polyvinylpyrrolidone, gelatin, medium chainfatty acid triglyceride, polyoxyethylene hydrogenated castor oil 60,saccharose, carboxymethylcellulose, corn starch, and inorganic salts.They may also contain other low-molecular-weight polypeptides; proteinssuch as serum albumin, gelatin, and immunoglobulin; and amino acids.When preparing aqueous solutions for injection, the anti-IL-6 receptorantibodies are dissolved, for example, in isotonic solutions containingphysiological saline, glucose, or other adjuvants. Adjuvants include,for example, D-sorbitol, D-mannose, D-mannitol, and sodium chloride.Furthermore, appropriate solubilizing agents, for example, alcohol(ethanol, and the like), polyalcohol (propylene glycol, PEG, and thelike), and non-ionic surfactants (polysorbate 80 and HCO-50) may becombined.

If necessary, the polypeptides may be encapsulated in microcapsules(microcapsules made of hydroxycellulose, gelatin, poly(methylmethacrylate), and the like), or made into a colloidal drug deliverysystem (liposomes, albumin microspheres, microemulsions, nanoparticles,nanocapsules, etc) (see, for example, “Remington's PharmaceuticalScience 16th edition”, Oslo Ed. (1980)). Moreover, methods for preparingagents as sustained-release agents are known, and these can be appliedto the polypeptides (Langer et al., J. Biomed. Mater. Res. (1981) 15:167-277; Langer, Chem. Tech. (1982) 12: 98-105; U.S. Pat. No. 3,773,919;European Patent Application (EP) No. 58,481; Sidman et al., Biopolymers(1983) 22:547-56; EP No.133,988). Furthermore, liquid volume forsubcutaneous administration can be increased by adding or mixinghyaluronidase to an agent (for example, see WO 2004/078140).

The pharmaceutical compositions of the present invention can beadministered both orally and parenterally, but are preferablyadministered parenterally. Specifically, the compositions areadministered to patients by injection or transdermally. Injectionsinclude, for example, systemic and local administrations by intravenous,intramuscular, or subcutaneous injection, or such. The compositions maybe locally injected at the site of treatment or in the periphery of thesite by intramuscular injection, in particular. Transdermal dosage formsinclude, for example, ointments, gel, cream, poultices, and patches,which can be administered locally or systemically. Furthermore,administration methods can be appropriately selected according to thepatient's age and symptoms. The administered dose can be selected, forexample, from the range of 0.0001 mg to 100 mg active ingredient per kgof body weight for each administration. Alternatively, when thecompositions are administered to human patients, for example, the activeingredient can be selected from the range of 0.001 to 1000 mg per kgbody weight for each patient. A single administration dose preferablycontains, for example, an antibody of the present invention at about0.01 to 50 mg/kg body weight. However, the dose of an antibody of thepresent invention is not limited to these doses.

Amino acids contained in the amino acid sequences in the presentinvention may be post-translationally modified (for example, themodification of an N-terminal glutamine into a pyroglutamic acid bypyroglutamylation is well-known to those skilled in the art). Naturally,such post-translationally modified amino acids are included in the aminoacid sequences in the present invention.

Further, sugar chains that are bound to the antibodies according to thepresent invention may be of any structure. A sugar chain at position 297(EU numbering) may be of any sugar chain structure (preferably afucosylated sugar chain), or no sugar chain may be bound (for example,this can be achieved by producing antibodies in Escherichia coli or byintroducing alteration so that no sugar chain binds to position 297, EUnumbering).

All prior art references cited herein are incorporated by reference intothis description.

EXAMPLES

Hereinbelow, the present invention will be specifically described withreference to the Examples, but it is not to be construed as beinglimited thereto.

Example 1 Identification of Mutation Sites in the Variable Regions forEnhancing the Affinity of TOCILIZUMAB for IL-6 Receptor

A library of CDR sequences into which mutations have been introduced wasconstructed and assayed to improve the affinity of TOCILIZUMAB (H chainWT-IgG1/SEQ ID NO: 53; L chain WT-kappa/SEQ ID NO: 54) for IL-6receptor. Screening of a library of CDR mutations revealed mutationsthat improve the affinity for IL-6 receptor. The mutations are shown inFIG. 1. A combination of these mutations yielded high-affinityTOCILIZUMAB such as RDC-23 (H chain RDC23H-IgG1/SEQ ID NO: 55; L chainRDC-23L-kappa/SEQ ID NO: 56). The affinity for soluble IL-6 receptor andbiological activity determined using BaF/gp130 were compared betweenRDC-23 and TOCILIZUMAB (see Reference Examples for the method).

The result of affinity measurement is shown in Table 1. The result ofbiological activity determination using BaF/gp130 (the finalconcentration of IL-6 was 30 ng/ml) is shown in FIG. 2. The resultsshowed that the affinity of RDC-23 was about 60 times higher, and theactivity expressed as concentration for 100% inhibition of BaF/gp130 wasabout 100 times higher when compared to TOCILIZUMAB.

TABLE 1 k_(a)(1/Ms) k_(d)(1/s) KD(M) TOCILIZUMAB 4.9E+05 2.0E−03 4.0E−09RDC-23 6.4E+05 4.3E−05 6.7E−11

Example 2 Identification of Mutations for Improving the Pharmacokineticsof TOCILIZUMAB via Reduction of its Isoelectric Point

To improve the pharmacokinetics of TOCILIZUMAB, investigation wascarried out to identify mutation sites that would decrease theisoelectric point of the variable regions without significantly reducingthe binding to the IL-6 receptor. Screening of mutation sites in thevariable regions, which were predicted based on a three-dimensionalstructure model of TOCILIZUMAB, revealed mutation sites that woulddecrease the isoelectric point of the variable regions withoutsignificantly reducing its binding to the IL-6 receptor. These are shownin FIG. 3. A combination of these mutations yielded TOCILIZUMAB withreduced isoelectric point including, for example, H53/L28 (H chainH53-IgG1/SEQ ID NO: 57; L chain L28-kappa/SEQ ID NO: 58). The affinityfor soluble IL-6 receptor, isoelectric point, pharmacokinetics in mice,and biological activity determined using BaF/gp130 were compared betweenH53/L28 and TOCILIZUMAB (see Reference Examples for the method).

The result of affinity measurement is shown in Table 2. The measurementresult for the biological activity obtained using BaF/gp130 (the finalconcentration of IL-6 was 30 ng/ml) is shown in FIG. 4. The resultsshowed that the affinity of H53/L28 was about six times higher and theactivity expressed as concentration for 100% inhibition of BaF/gp130 wasabout several times higher when compared to TOCILIZUMAB.

TABLE 2 k_(a)(1/Ms) k_(d)(1/s) KD(M) TOCILIZUMAB 4.9E+05 2.0E−03 4.0E−09H53/L28 7.6E+05 5.2E−04 6.8E−10

The result of isoelectric point determination by isoelectric pointelectrophoresis known to those skilled in the art showed that theisoelectric points of TOCILIZUMAB and H53/L28 were about 9.3 and 6.5 to6.7, respectively. Thus, the isoelectric point of H53/L28 was reduced byabout 2.7 when compared to TOCILIZUMAB. Furthermore, the theoreticalisoelectric point of the VH/VL variable regions was calculated usingGENETYX (GENETYX CORPORATION). The result showed that the theoreticalisoelectric points of TOCILIZUMAB and H53/L28 were 9.20 and 4.52,respectively. Thus, the isoelectric point of H53/L28 was reduced byabout 4.7 when compared to TOCILIZUMAB.

To assess the pharmacokinetics of the altered antibody H53/L28 which hasa reduced isoelectric point, the pharmacokinetics of TOCILIZUMAB andH53/L28 in normal mice were compared. A single dose of TOCILIZUMAB orH53/L28 was intravenously (IV) or subcutaneously (SC) administered at 1mg/kg to mice (C57BL/6J; Charles River Japan, Inc.) to evaluate the timecourse of plasma concentration. The time courses of plasma concentrationfor TOCILIZUMAB and H53/L28 after intravenous administration orsubcutaneous administration are shown in FIGS. 5 and 6, respectively.Pharmacokinetic parameters (clearance (CL) and half-life (T½)) obtainedusing WinNonlin (Pharsight) are shown in Table 3. The plasma half-life(T½) of H53/L28 after intravenous administration was prolonged to about1.3 times that of TOCILIZUMAB, while the clearance was reduced by about1.7 times. T½ of H53/L28 after subcutaneous administration was increasedto about twice that of TOCILIZUMAB, while the clearance was reduced byabout 2.1 times. Thus, it was found that the pharmacokinetics could besignificantly improved by reducing the isoelectric point of TOCILIZUMABthrough amino acid substitution.

TABLE 3 IV SC CL T½ CL/F T½ mL/h/kg day mL/h/kg day TOCILIZUMAB 0.17718.5 0.18 14.7 H53/L28 0.102 23.5 0.086 29.7

Example 3 Identification of Mutation Sites that Reduce theImmunogenicity of TOCILIZUMAB

Identification of Mutations that Reduce the Immunogenicity Risk ofT-cell Epitopes Present in the variable Regions

T-cell epitopes present in the variable-region sequence of TOCILIZUMABwere analyzed using TEPITOPE (Methods. 2004 December; 34(4):468-75). Asa result, the L-chain CDR2 was predicted to have many T-cell epitopesthat would bind to HLA (i.e. to have a sequence with a highimmunogenicity risk). Thus, TEPITOPE analysis was carried out to examineamino acid substitutions that would reduce the immunogenicity risk ofthe L-chain CDR2 without decreasing the stability, binding activity, orneutralizing activity.

As described below, the screening result demonstrated that theimmunogenicity risk can be reduced without decreasing the stability,binding activity, or neutralizing activity by substituting the threonineat L51 (Kabat's numbering; Kabat EA et al., (1991) Sequences of Proteinsof Immunological Interest, NIH)) of the L chain CDR2 (SEQ ID NO: 59) ofTOCILIZUMAB with glycine, and the arginine at L53 with glutamic acid(SEQ ID NO: 60).

-   -   TOCILIZUMAB L-chain CDR2 (SEQ ID NO: 59)    -   TOCILIZUMAB L-chain CDR2 with T-cell epitopes removed (SEQ ID        NO: 60)

Example 4 Reduction of Immunogenicity Risk by Full Humanization of theVariable Region Framework Sequences of TOCILIZUMAB

In the process of TOCILIZUMAB humanization, some mouse sequences remainin the framework sequence to maintain binding activity (Cancer Res. 1993Feb. 15; 53(4):851-6). These sequences are H27, H28, H29, and H30 in theH-chain FR1, and H71 in the H-chain FR3 (Kabat's numbering; Kabat E A etal., (1991) Sequences of Proteins of Immunological Interest, NIH)) ofthe variable region sequence of TOCILIZUMAB. The mouse sequences thatremained are a potential cause of increased immunogenicity risk. Thus,it was assessed whether the framework sequence could be fully humanizedto further reduce the immunogenicity risk of TOCILIZUMAB.

The result showed that the entire framework of TOCILIZUMAB could becompletely humanized without decreasing the stability, binding activity,or neutralizing activity, by substituting the H-chain FR1 (SEQ ID NO:61) of TOCILIZUMAB with the humanized H-chain FR1-A (SEQ ID NO: 62)shown below, and substituting the-H chain FR3 (SEQ ID NO: 63) with thehumanized H chain FR3 (SEQ ID NO: 64) shown below.

-   -   TOCILIZUMAB H chain FR1 (SEQ ID NO: 61)    -   Humanized H chain FR1-A (SEQ ID NO: 62) (derived from germline        IMGT hVH_(—)4)    -   TOCILIZUMAB H chain FR3 (SEQ ID NO: 63)    -   Humanized H chain FR3 (SEQ ID NO: 64) (derived from Mol.        Immunol. 2007, 44(4):412-422)

Example 5 Identification of Mutation Sites to Improve thePharmacokinetics Based on pH-Dependent Binding of TOCILIZUMAB to theIL-6 Receptor

One of the methods for improving the pharmacokinetics of TOCILIZUMAB isto improve the molecule such that a single molecule of TOCILIZUMAB wouldrepeatedly bind and neutralize several molecules of the IL-6 receptor.It is assumed that after binding to membrane-type IL-6 receptor,TOCILIZUMAB is taken up into intracellular endosomes via internalizationwhile bound to membrane-type IL-6 receptor, then transferred intolysosomes while bound to membrane-type IL-6 receptor, and becomesdegraded by lysosomes. Specifically, one molecule of TOCILIZUMABtypically binds to one or two molecules of membrane-type IL-6 receptor(in a monovalent or divalent manner) and is degraded in lysosomes afterinternalization. Therefore, one molecule of TOCILIZUMAB can only bindand neutralize one or two molecules of membrane-type IL-6 receptor.

Thus, the present inventors thought that if it were possible to createTOCILIZUMAB that binds in a pH-dependent manner, in which the binding ofTOCILIZUMAB is maintained under neutral conditions but significantlyreduced under acidic conditions, TOCILIZUMAB which binds in apH-dependent manner could dissociate from membrane-type IL-6 receptor(antigen) in the endosomes and return to the plasma by binding to FcRnpresent in the endosomes, as illustrated in FIG. 7. Once returned to theplasma, TOCILIZUMAB which binds in a pH-dependent manner could againbind to membrane-type IL-6 receptor. By repeating this binding in theplasma and dissociation in the endosomes, it is thought that onemolecule of TOCILIZUMAB can repeatedly bind/neutralize several moleculesof the IL-6 receptor. Thus, TOCILIZUMAB which binds in a pH-dependentmanner is assumed to have improved pharmacokinetics as compared toTOCILIZUMAB.

For TOCILIZUMAB to dissociate from the IL-6 receptor under the acidiccondition in the endosome, the binding must be significantly weakenedunder the acidic condition as compared to under the neutral condition.On the cell surface, strong IL-6 receptor binding is required forneutralization; therefore, at pH 7.4 which is the cell surface pH, theantibody must bind to the IL-6 receptor as strongly as or more stronglythan TOCILIZUMAB. It has been reported that the endosomal pH isgenerally 5.5 to 6.0 (Nat Rev Mol Cell Biol. 2004 February;5(2):121-32). Thus, if TOCILIZUMAB which binds in a pH-dependent manneris modified to weakly bind to the IL-6 receptor at pH 5.5 to 6.0, it canbe predicted to dissociate from the IL-6 receptor under the acidiccondition in the endosomes. Specifically, if TOCILIZUMAB which binds ina pH-dependent manner is improved to strongly bind to the IL-6 receptorat pH 7.4, which is the cell surface pH, and to weakly bind to IL-6receptor at pH 5.5 to 6.0, which is the endosomal pH, one molecule ofTOCILIZUMAB can bind and neutralize several molecules of the IL-6receptor, and the pharmacokinetics can therefore be improved.

A possible method for conferring pH dependence on the binding ofTOCILIZUMAB to the IL-6 receptor is to introduce histidine residues intothe variable region of TOCILIZUMAB, since the pKa of a histidine residueis about 6.0 to 6.5, and its state of proton dissociation changesbetween neutral (pH 7.4) and acidic (pH 5.5 to 6.0) conditions. Thus,screening was carried out to identify sites for histidine introductionin the variable regions based on a three-dimensional structure model ofTOCILIZUMAB. Furthermore, selected variable region sequences ofTOCILIZUMAB were randomly substituted with histidine to design a libraryfor screening. The screening was carried out using the binding to theIL-6 receptor at pH 7.4 and dissociation from the IL-6 receptor, or thereduction of affinity at pH 5.5 to 5.8 as an index.

As a result, the present inventors discovered mutation sites that conferthe binding of TOCILIZUMAB to the IL-6 receptor with pH dependency (theproperty to bind at pH 7.4 and dissociate at pH 5.8). These are shown inFIG. 8. In FIG. 8, the substitution of tyrosine at H27 to histidine is amutation in the H-chain FR1, not in the CDR. However, as described inEur. J. Immunol. (1992) 22: 1719-1728, a sequence with histidine at H27is a human sequence (SEQ ID NO: 65). Thus, the antibody can becompletely humanized by using the following framework in combinationwith Example 4.

-   -   Humanized H-chain FR1-B (SEQ ID NO: 65)

A combination of mutations including, for example, H3pI/L73 (H chainH3pI-IgG1/SEQ ID NO: 66; L chain L73-kappa/SEQ ID NO: 67) can yieldTOCILIZUMAB with pH-dependent binding properties. H3pI/L73 andTOCILIZUMAB were compared for their affinity towards soluble IL-6receptor at pH 7.4, rate of dissociation from membrane-type IL-6receptor at pH 7.4 and pH 5.8, biological activity using BaF/gp130, andpharmacokinetics in cynomolgus monkey and human IL-6 receptor transgenicmice (see Reference Examples for the method).

The result of affinity assay for soluble IL-6 receptor at pH 7.4 isshown in Table 4. The assay result for the biological activity obtainedusing BaF/gp130 (final IL-6 concentration of 30 ng/ml) is shown in FIG.9. These results showed that H3pI/L73 is comparable to TOCILIZUMAB interms of affinity for soluble IL-6 receptor at pH 7.4 and activity onBaF/gp130.

TABLE 4 k_(a)(1/Ms) k_(d)(1/s) KD(M) TOCILIZUMAB 5.1E+05 1.0E−03 2.1E−09H3pI/L73 5.4E+05 7.4E−04 1.4E−09

The measurement result for the rate of dissociation of TOCILIZUMAB orH3pI/L73 from membrane-type IL-6 receptor at pH 7.4 and pH 5.8 is shownin Table 5. As compared to TOCILIZUMAB, the dissociation rate ofH3pI/L73 at pH 5.8 was faster and the pH dependence of the rate ofdissociation from membrane-type IL-6 receptor was increased by about 2.6times.

TABLE 5 pH 7.4 pH 5.8 k_(d(pH 5.8))/k_(d(pH 7.4)) k_(d)(1/s) k_(d)(1/s)pH DEPENDENCY TOCILIZUMAB 2.5E−04 2.5E−04 1.00 H3pI/L73 2.6E−04 6.7E−042.59

A single dose of TOCILIZUMAB or H3pI/L73 was intravenously administeredat 1 mg/kg to cynomolgus monkeys to assess the time course of plasmaconcentration. The plasma concentration time courses of TOCILIZUMAB orH3pI/L73 after intravenous administration are shown in FIG. 10. Theresult showed that the pharmacokinetics of H3pI/L73 in cynomolgusmonkeys was significantly improved as compared to TOCILIZUMAB.

A single dose of TOCILIZUMAB or H3pI/L73 was intravenously administeredat 25 mg/kg to human IL-6 receptor transgenic mice (hIL-6R tg mice; ProcNatl Acad Sci USA. 1995 May 23; 92(11):4862-6) to assess the time courseof plasma concentration. The plasma concentration time courses ofTOCILIZUMAB or H3pI/L73 after intravenous administration are shown inFIG. 11. The result showed that the pharmacokinetics of H3pI/L73 inhuman IL-6 receptor transgenic mice was significantly improved ascompared to TOCILIZUMAB.

H3pI/L73, which is a TOCILIZUMAB with pH-dependent binding properties,showed significantly improved pharmacokinetics in cynomolgus monkeys andhuman IL-6 receptor transgenic mice when compared to TOCILIZUMAB. Thissuggests that it is possible to bind to and neutralize several moleculesof the IL-6 receptor with one single molecule, by conferring theproperty of binding an antigen at pH 7.4 and dissociating from theantigen at pH 5.8. It was also considered that the pharmacokineticscould be further improved by conferring IL-6 receptor binding with amore pronounced pH dependence than that of H3pI/L73.

Example 6 Optimization of the TOCILIZUMAB Constant Region

Reduction of the Heterogeneity of TOCILIZUMAB H-Chain C Terminus

For heterogeneity of the H-chain C-terminal sequences of an IgGantibody, deletion of C-terminal amino acid lysine residue, andamidation of the C-terminal carboxyl group due to deletion of both ofthe two C-terminal amino acids, glycine and lysine, have been reported(Anal Biochem. 2007 Jan 1; 360(1):75-83). Also in TOCILIZUMAB, the majorcomponent is a sequence in which the C-terminal amino acid lysine in thenucleotide sequence is deleted by post-translational modification;however, sub-components in which the lysine remains and sub-componentsin which the C-terminal carboxyl group is amidated due to deletion ofboth glycine and lysine also exist as heterogeneity. It is not easy andwould be more costly to manufacture them as a pharmaceutical inlarge-scale while maintaining the objective substances/relatedsubstances related heterogeneity between productions. If possible, it isdesirable to be single substances, and to have reduced heterogeneitywhen developing antibodies as pharmaceuticals. Thus, it is preferablethat the H-chain C-terminal heterogeneity is absent when developingantibodies as pharmaceuticals.

The C-terminal amino acid was altered to reduce the C-terminal aminoacid heterogeneity. The result showed that the C-terminus-derivedheterogeneity can be prevented by pre-deleting from the nucleotidesequence, the lysine and glycine residues at the C terminus of theH-chain constant region of TOCILIZUMAB. TOCILIZUMAB, TOCILIZUMAB thatlacks the C-terminal lysine residue (TOCILIZUMABAK: H chainWT-IgG1ΔK/SEQ ID NO: 68; L chain WT-kappa/SEQ ID NO: 54), andTOCILIZUMAB that lacks the C-terminal lysine and glycine residues(TOCILIZUMABAGK: H chain WT-IgG1ΔGK/SEQ ID NO: 69; L chain WT-kappa/SEQID NO: 54) were assessed for heterogeneity by cation exchangechromatography. The ProPac WCX-10, 4×250 mm (Dionex) column was used;and mobile phase A was 25 mmol/L MES/NaOH (pH 6.1) and mobile phase Bwas 25 mmol/L MES/NaOH, 250 mmol/L NaCl (pH 6.1). Appropriate flow rateand gradient were used. The assessment result obtained by cationexchange chromatography is shown in FIG. 12. The result showed that theC-terminal amino acid heterogeneity can be reduced by pre-deleting fromthe nucleotide sequence both the lysine and glycine residues at the Cterminus of the H-chain constant region, but not by pre-deleting onlythe lysine residue at the C terminus of the H-chain constant region. Allof the C-terminal sequences of the constant region of human antibodiesIgG1, IgG2, and IgG4 contain lysine and glycine at positions 447 and446, respectively, according to EU numbering (see Sequences of proteinsof immunological interest, NIH Publication No.91-3242). Therefore, themethod for reducing the C-terminal amino acid heterogeneity found in thepresent study is expected to be also applicable to IgG2 and IgG4constant regions and variants thereof.

Reduction of Disulfide Bond-Derived Heterogeneity in IgG2 IsotypeTOCILIZUMAB

The isotype of TOCILIZUMAB is IgG1. Since TOCILIZUMAB is a neutralizingantibody, binding to the Fcγ receptor can be unfavorable in view ofimmunogenicity and adverse effects. A possible method for lowering theFcγ receptor binding is to convert the isotype of the IgG antibody fromIgG1 to IgG2 or IgG4 (Ann Hematol. 1998 June; 76(6):231-48). From theviewpoint of Fcγ receptor I binding and pharmacokinetics, IgG2 wasconsidered to be more desirable than IgG4 (Nat Biotechnol. 2007 Dec;25(12):1369-72). Meanwhile, physicochemical properties of proteins, inparticular, homogeneity and stability are very important when developingantibodies as pharmaceuticals. The IgG2 isotype has been reported tohave very high heterogeneity due to the disulfide bonds in the hingeregion (J Biol Chem. 2008 Jun. 6; 283(23):16206-15). It is not easy andwould be more costly to manufacture them as pharmaceutical inlarge-scale while maintaining the objective substances/relatedsubstances related heterogeneity derived from disulfide bonds betweenproductions. Thus, single substances are desirable as much as possible.Thus, when developing IgG2 isotype antibodies into pharmaceuticals, itis preferable to reduce the heterogeneity derived from disulfide bondswithout lowering the stability.

For the purpose of reducing the heterogeneity of the IgG2 isotype,various variants were assessed. As a result, it was found thatheterogeneity could be reduced without decreasing the stability usingthe WT-SKSC constant region (SEQ ID NO: 70), in which of the IgG2constant region sequences, the cysteine residue at position 131 and thearginine residue at position 133 (EU numbering) in the H-chain CH1domain were substituted to serine and lysine, respectively, and thecysteine residue at position 219 (EU numbering) in the H-chain upperhinge was substituted to serine. TOCILIZUMAB-IgG1 (H chain WT-IgG1/SEQID NO: 53; L chain WT-kappa/SEQ ID NO: 54), TOCILIZUMAB-IgG2 (H chainWT-IgG2/SEQ ID NO: 71; L chain WT-kappa/SEQ ID NO: 54), andTOCILIZUMAB-SKSC (H chain WT-SKSC/SEQ ID NO: 70; L chain WT-kappa/SEQ IDNO: 54) were prepared and assessed for heterogeneity and stability. Theheterogeneity was assessed by cation exchange chromatography. The ProPacWCX-10 (Dionex) column was used; and mobile phase A was 20 mM SodiumAcetate (pH 5.0) and mobile phase B was 20 mM Sodium Acetate, 1 M NaCl(pH 5.0). Appropriate flow rate and gradient were used. The assessmentresult obtained by cation exchange chromatography is shown in FIG. 13.The stability was assessed based on the intermediate temperature inthermal denaturation (Tm value) determined by differential scanningcalorimetry (DSC) (VP-DSC; Microcal). The result of DSC measurement in20 mM sodium acetate, 150 mM NaCl, pH 6.0 and the Tm value of the Fabdomain are shown in FIG. 14.

The result showed that the heterogeneity was markedly increased inTOCILIZUMAB-IgG2 as compared to TOCILIZUMAB-IgG1; however, theheterogeneity could be significantly reduced by conversion toTOCILIZUMAB-SKSC. Furthermore, when compared to TOCILIZUMAB-IgG1, theDSC of TOCILIZUMAB-IgG2 gave a shoulder peak (Fab*) component with lowstability, i.e., low Tm, in the thermal denaturation peaks of the Fabdomain, which is assumed to be due to a heterogeneous component.However, when converted to TOCILIZUMAB-SKSC, the shoulder peak (low Tm),which is thought to be due to a heterogeneous component, disappeared,and the Tm value was about 94° C., which was equivalent to that of theFab domain of TOCILIZUMAB-IgG1 and TOCILIZUMAB-IgG2. Thus,TOCILIZUMAB-SKSC was revealed to have high stability.

Identification of Pharmacokinetics-Improving Mutation Sites in theConstant Region of TOCILIZUMAB

As described above, starting from IgG1, which is the isotype ofTOCILIZUMAB, reduction of the C-terminal heterogeneity and reduction ofheterogeneity of antibodies with IgG2 isotype constant regions whilereducing the binding to the Fcγ receptor and maintaining the highstability can be achieved. Moreover, it is preferred that the constantregion also has superior pharmacokinetics than IgG1, which is theisotype of TOCILIZUMAB.

In order to find constant regions having a superior plasma half-lifethan antibodies with IgG1-isotype constant regions, screening wascarried out to identify mutation sites for improving thepharmacokinetics of TOCILIZUMAB-SKSC which has high stability andreduced heterogeneity related to antibodies with IgG2-isotype constantregions as mentioned above. As a result, WT-M58 (SEQ ID NO: 72 (aminoacid sequence)) was discovered, in which, as compared to WT-SKSC, theglutamic acid at position 137, EU numbering is substituted to glycine,the serine at position 138 is substituted to glycine, the histidine atposition 268 is substituted to glutamine, the arginine at position 355is substituted to glutamine, the glutamine at position 419 issubstituted to glutamic acid, and in which the glycine at position 446and the lysine at position 447 is deleted to reduce the heterogeneity ofthe H-chain C terminus. In addition, WT-M44 (SEQ ID NO: 73 (amino acidsequence)) was prepared to have substitution of asparagine at position434 to alanine, relative to IgG1. Furthermore, WT-M83 (SEQ ID NO: 74(amino acid sequence)) was produced by deleting glycine at position 446and lysine at position 447 from M44 to reduce the heterogeneity of theH-chain C-terminus. In addition, WT-M73 (SEQ ID NO: 75 (amino acidsequence)) was produced by substituting asparagine at position 434 withalanine in WT-M58.

TOCILIZUMAB-M44 (H chain WT-M44/SEQ ID NO: 73; L chain WT-kappa/SEQ IDNO: 54), TOCILIZUMAB-M58 (H chain WT-M58/SEQ ID NO: 72; L chainWT-kappa/SEQ ID NO: 54), and TOCILIZUMAB-M73 (H chain WT-M73/SEQ ID NO:75; L chain WT-kappa/SEQ ID NO: 54) were prepared and assessed for theiraffinity towards human FcRn and pharmacokinetics using human FcRntransgenic mice (see Reference Examples for the method).

The binding of TOCILIZUMAB-IgG1, TOCILIZUMAB-M44, TOCILIZUMAB-M58, andTOCILIZUMAB-M73 to human FcRn was assessed using Biacore. As shown inTable 6, the binding of TOCILIZUMAB-M44, TOCILIZUMAB-M58, andTOCILIZUMAB-M73 was about 2.7 times, 1.4 times, and 3.8 times superiorthan that of TOCILIZUMAB-IgG1, respectively.

TABLE 6 KD(μM) TOCILIZUMAB-IgG1 1.62 TOCILIZUMAB-M44 0.59TOCILIZUMAB-M58 1.17 TOCILIZUMAB-M73 0.42

TOCILIZUMAB-IgG1, TOCILIZUMAB-M44, TOCILIZUMAB-M58, and TOCILIZUMAB-M73were assessed for their pharmacokinetics in human FcRn transgenic mice.The result is shown in FIG. 15. When compared to TOCILIZUMAB-IgG1, allof TOCILIZUMAB-M44, TOCILIZUMAB-M58, and TOCILIZUMAB-M73 were found toexhibit improved pharmacokinetics, as shown in FIG. 15. The effect ofimproving the pharmacokinetics correlated with the ability to bind tohuman FcRn. In particular, the concentration of TOCILIZUMAB-M73 inplasma after 28 days was improved by about 16 times as compared toTOCILIZUMAB-IgG1. Thus, antibodies having the constant region of M73were also assumed to have significantly improved pharmacokinetics inhumans as compared to antibodies having the IgG1 constant region.

Example 7 Preparation of Fully Humanized IL-6 Receptor Antibodies withImproved PK/PD

TOCILIZUMAB variants were prepared by combining multiple mutations inthe variable and constant regions of TOCILIZUMAB found in the examplesabove. Fully humanized IL-6 receptor antibodies discovered from variousscreenings were: Fv3-M73 (H chain VH4-M73/SEQ ID NO: 25; L chainVL1-kappa/SEQ ID NO: 28), Fv4-M73 (H chain VH3-M73/SEQ ID NO: 26; Lchain VL3-kappa/SEQ ID NO: 29), and Fv5-M83 (H chain VH5-M83/SEQ ID NO:27; L chain VL5-kappa/SEQ ID NO: 30).

The affinities of prepared Fv3-M73, Fv4-M73, and Fv5-M83 against IL-6receptor were compared to that of TOCILIZUMAB (see Reference Example formethod). The affinities of these antibodies for the soluble IL-6receptor determined at pH 7.4 are shown in Table 7. Furthermore, theirBaF/gp130-neutralizing activities were compared to those of TOCILIZUMABand the control (the known high affinity anti-IL-6 receptor antibodydescribed in Reference Example, and VQ8F11-21 hIgG1 described in US2007/0280945) (see Reference Example for method). The results obtainedby determining the biological activities of these antibodies usingBaF/gp130 are shown in FIG. 16 (TOCILIZUMAB, the control, and Fv5-M83with a final IL-6 concentration of 300 ng/ml) and FIG. 17 (TOCILIZUMAB,Fv3-M73, and Fv4-M73 with a final IL-6 concentration of 30 ng/ml). Asshown in Table 7, Fv3-M73 and Fv4-M73 have about two to three timeshigher affinity than TOCILIZUMAB, while Fv5-M83 exhibits about 100 timeshigher affinity than TOCILIZUMAB (since it was difficult to measure theaffinity of Fv5-M83, instead the affinity was determined using Fv5-IgG1(H chain VH5-IgG1/SEQ ID NO: 76; L chain VL5-kappa/SEQ ID NO: 30), whichhas an IgG1-type constant region; the constant region is generallythought to have no effect on affinity). As shown in FIG. 17, Fv3-M73 andFv4-M73 exhibit slightly stronger activities than TOCILIZUMAB. As shownin FIG. 16, Fv5-M83 has a very strong activity, which is more than 100times greater than that of TOCILIZUMAB in terms of 50% inhibitoryconcentration. Fv5-M83 also exhibits about 10 times higher neutralizingactivity in terms of 50% inhibitory concentration than the control (theknown high-affinity anti-IL-6 receptor antibody).

TABLE 7 k_(a)(1/Ms) k_(d)(1/s) KD(M) TOCILIZUMAB 4.0E+05 1.1E−03 2.7E−09Fv3-M73 8.5E+05 8.7E−04 1.0E−09 Fv4-M73 7.5E+05 1.0E−03 1.4E−09 Fv5-M831.1E+06 2.8E−05 2.5E−11

The rates of dissociation of TOCILIZUMAB, Fv3-M73, and Fv4-M73 frommembrane-type IL-6 receptor at pH 7.4 and 5.8 were determined. Asdemonstrated by the result shown in Table 8 (see Reference Example formethod), the pH dependency of the dissociation rate of Fv3-M73 andFv4-M73 from membrane-type IL-6 receptor was about 11 times and 10 timesimproved, respectively, as compared to TOCILIZUMAB. The considerableimprovement of the pH dependency of the dissociation rate relative toH3pI/L73 described in Example 5 suggested that when compared toH3pI/L73, pharmacokinetics of Fv3-M73 and Fv4-M73 would be significantlyimproved.

TABLE 8 pH 7.4 pH 5.8 k_(d(pH 5.8))/k_(d(pH 7.4)) k_(d)(1/s) k_(d)(1/s)pH DEPENDENCY TOCILIZUMAB 2.5E−04 2.5E−04 1.00 Fv3-M73 4.9E−04 5.3E−0310.88 Fv4-M73 5.1E−04 5.1E−03 10.06

The isoelectric points of TOCILIZUMAB, the control, Fv3-M73, Fv4-M73,and Fv5-M83 were determined by isoelectric focusing electrophoresisusing a method known to those skilled in the art. The result showed thatthe isoelectric point was about 9.3 for TOCILIZUMAB; about 8.4 to 8.5for the control; about 5.7 to 5.8 for Fv3-M73; about 5.6 to 5.7 forFv4-M73; and 5.4 to 5.5 for Fv5-M83. Thus, each antibody had asignificantly lowered isoelectric point when compared to TOCILIZUMAB andthe control. Furthermore, the theoretical isoelectric point of thevariable regions VH/VL was calculated by GENETYX (GENETYX CORPORATION).The result showed that the theoretical isoelectric point was 9.20 forTOCILIZUMAB; 7.79 for the control; 5.49 for Fv3-M73; 5.01 for Fv4-M73;and 4.27 for Fv5-M83. Thus, each antibody had a significantly loweredisoelectric point when compared to TOCILIZUMAB and the control. Since itwas shown in Example 2 that pharmacokinetics is improved by reducing theisoelectric point, the pharmacokinetics of Fv3-M73, Fv4-M73, and Fv5-M83was thought to be improved when compared to TOCILIZUMAB and the control.

T-cell epitopes in the variable region sequence of TOCILIZUMAB, Fv3-M73,Fv4-M73, or Fv5-M83 were analyzed using TEPITOPE (Methods. 2004December; 34(4):468-75). As a result, TOCILIZUMAB was predicted to haveT-cell epitopes, of which many could bind to HLA, as shown in Example 3.In contrast, the number of sequences that were predicted to bind toT-cell epitopes was significantly reduced in Fv3-M73, Fv4-M73, andFv5-M83. In addition, the framework of Fv3-M73, Fv4-M73, or Fv5-M83 hasno mouse sequence and is thus fully humanized. These suggest thepossibility that immunogenicity risk is significantly reduced inFv3-M73, Fv4-M73, and Fv5-M83 when compared to TOCILIZUMAB.

Example 8 PK/PD Test of Fully Humanized IL-6 Receptor Antibodies inMonkeys

Each of TOCILIZUMAB, the control, Fv3-M73, Fv4-M73, and Fv5-M83 wasintravenously administered once at a dose of 1 mg/kg to cynomolgusmonkeys to assess their time course of plasma concentration (seeReference Example for method). The plasma concentration time courses ofTOCILIZUMAB, Fv3-M73, Fv4-M73, and Fv5-M83 after intravenousadministration are shown in FIG. 18. The result showed that each ofFv3-M73, Fv4-M73, and Fv5-M83 exhibited significantly improvedpharmacokinetics in cynomolgus monkeys when compared to TOCILIZUMAB andthe control. Of them, Fv3-M73 and Fv4-M73 exhibited highly improvedpharmacokinetics when compared to TOCILIZUMAB.

The efficacy of each antibody to neutralize membrane-type cynomolgusmonkey IL-6 receptor was assessed. Cynomolgus monkey IL-6 wasadministered subcutaneously in the lower back at 5 μg/kg every day fromDay 6 to Day 18 after antibody administration (Day 3 to Day 10 forTOCILIZUMAB), and the CRP concentration in each animal was determined 24hours later (see Reference Example for method). The time course of CRPconcentration after administration of each antibody is shown in FIG. 19.To assess the efficacy of each antibody to neutralize soluble cynomolgusmonkey IL-6 receptor, the plasma concentration of free solublecynomolgus monkey IL-6 receptor in the cynomolgus monkeys was determinedand the percentages of free soluble IL-6 receptor were calculated (seeReference Example for method). The time course of percentage of freesoluble IL-6 receptor after administration of each antibody is shown inFIG. 20.

Each of Fv3-M73, Fv4-M73, and Fv5-M83 neutralized membrane-typecynomolgus monkey IL-6 receptor in a more sustainable way, andsuppressed the increase of CRP over a longer period when compared toTOCILIZUMAB and the control (the known high-affinity anti-IL-6 receptorantibody). Furthermore, each of Fv3-M73, Fv4-M73, and Fv5-M83neutralized soluble cynomolgus monkey IL-6 receptor in a moresustainable way, and suppressed the increase of free soluble cynomolgusmonkey IL-6 receptor over a longer period when compared to TOCILIZUMABand the control. These findings demonstrate that all of Fv3-M73,Fv4-M73, and Fv5-M83 are superior in sustaining the neutralization ofmembrane-type and soluble IL-6 receptors than TOCILIZUMAB and thecontrol. Of them, Fv3-M73 and Fv4-M73 are remarkably superior insustaining the neutralization. Meanwhile, Fv5-M83 suppressed CRP andfree soluble cynomolgus monkey IL-6 receptor more strongly than Fv3-M73and Fv4-M73. Thus, Fv5-M83 is considered to be stronger than Fv3-M73,Fv4-M73, and the control (the known high-affinity anti-IL-6 receptorantibody) in neutralizing membrane-type and soluble IL-6 receptors. Itwas considered that results in in vivo of cynomolgus monkeys reflect thestronger affinity of Fv5-M83 for IL-6 receptor and stronger biologicalactivity of Fv5-M83 in the BaF/gp130 assay system relative to thecontrol.

These findings suggest that Fv3-M73 and Fv4-M73 are highly superior insustaining their activities as an anti-IL-6 receptor-neutralizingantibody when compared to TOCILIZUMAB and the control, and thus enableto significantly reduce the dosage and frequency of administration.Furthermore, Fv5-M83 was demonstrated to be remarkably superior in termsof the strength of activity as an anti-IL-6 receptor-neutralizingantibody as well as sustaining their activity. Thus, Fv3-M73, Fv4-M73,and Fv5-M83 are expected to be useful as pharmaceutical IL-6antagonists.

Example 9

Monocyte chemoattractant protein (MCP)-1 is known to be involved incellular invasion of monocytes, T cells, NK cells, and basophils. MCP-1has been reported to be highly expressed in synovial tissues/synovialfluid of RA patients (J. Clin. Invest., September 1992, 90(3):772-779)and is thought to be involved in the pathological condition of RA(Inflamm. Allergy Drug Targets, March 2008, 7(1):53-66).

VEGF is a potent angiogenic factor and is known to be produced, forexample, by macrophages, fibroblasts, and synovial cells in the synovialmembrane of RA patients (J. Rheumatol., September 1995,22(9):1624-1630). Moreover, the VEGF level in the serum of RA patientscorrelates with disease activity and radiographic progression (ArthritisRheum., June 2003, 48(6):1521-1529; and Arthritis Rheum., September2001, 44(9):2055-2064) and the VEGF level in the serum decreases bytreating RA patients with the anti-IL-6R antibody TOCILIZUMAB;therefore, VEGF is also considered to play an important role in thepathological condition of RA (Mod. Rheumatol. 2009, 19(1):12-19; andMediators Inflamm. 2008, 2008:129873).

Thus, whether TOCILIZUMAB and Fv4-M73 can inhibit MCP-1 and VEGFproductions from human RA patient-derived synovial cells which occurfrom sIL-6R and IL-6 stimulation was examined.

Human RA patient-derived synovial cells (TOYOBO) were plated onto 96well plates in 5% FCS-containing IMDM medium at 2×10⁴ cells/0.05mL/well, and placed for 90 minutes in a CO₂ incubator (37° C., 5% CO₂).0.05 mL of TOCILIZUMAB and Fv4-M73 diluted to appropriate concentrationswere added, the plates were left still for 15 minutes, then 0.05 mL ofs0oluble IL-6 receptor (SR344: prepared according to the methoddescribed in Reference Examples) were added. The plates were furtherleft still for 30 minutes, and 0.05 mL of IL-6 (TORAY) were furtheradded (the final concentrations of soluble IL-6 receptor and IL-6 were50 ng/mL for each). After two days of culture, the culture supernatantswere collected, and the MCP-1 and VEGF concentrations in the culturesupernatants were measured using ELISA kit (Biosource and PierceBiotechnology). The results are shown in FIGS. 21 and 22. TOCILIZUMABand Fv4-M73 inhibited MCP-1 and VEGF production from human RApatient-derived synovial cells following soluble IL-6 receptor and IL-6stimulation in a concentration-dependent manner.

Accordingly, the persistence of the effect of Fv4-M73 as an anti-IL-6receptor neutralizing antibody (the effect of binding to the IL-6receptor and blocking the signals of the membrane-type IL6 receptor andsoluble IL-6 receptor) is significantly superior as compared toTOCILIZUMAB, the administration frequency and dose can be greatlyreduced as compared to TOCILIZUMAB, and furthermore, Fv4-M73 inhibitsMCP-1 and VEGF production from human RA patient-derived synovial cells.Therefore, Fv4-M73 was shown to be a very effective therapeutic agentagainst RA.

Reference Examples

Preparation of Soluble Recombinant Human IL-6 Receptor

Soluble recombinant human IL-6 receptor of the human IL-6 receptor,which is the antigen, was produced as described below. A CHO cell lineconstitutively expressing a soluble human IL-6 receptor containing asequence from the N-terminal 1st to 344th amino acids reported in J.Biochem. (1990) 108, 673-676 (Yamasaki et al., Science (1988) 241,825-828 (GenBank #X12830)) was generated. Soluble human IL-6 receptorwas purified from culture supernatant of CHO cells expressing SR344 bythree column chromatographies: Blue Sepharose 6 FF columnchromatography, affinity chromatography using a column immobilized withan antibody specific to SR344, and gel filtration column chromatography.The fraction eluted as the main peak was used as the final purifiedsample.

Preparation of Soluble Recombinant Cynomolgus Monkey IL-6 Receptor(cIL-6R)

Oligo-DNA primers were prepared based on the disclosed gene sequence forRhesus monkey IL-6 receptor (Birney et al., Ensembl 2006, Nucleic AcidsRes. 2006 Jan. 1; 34 (Database issue):D556-61). A DNA fragment encodingthe whole cynomolgus monkey IL-6 receptor gene was prepared by PCR usingthe primers, and as a template, cDNA prepared from the pancreas ofcynomolgus monkey. The resulting DNA fragment was inserted into amammalian cell expression vector, and a stable expression CHO line(cyno.sIL-6R-producing CHO cell line) was prepared using the vector. Theculture medium of cyno.sIL-6R-producing CHO cells was purified using aHisTrap column (GE Healthcare Bioscience) and then concentrated withAmicon Ultra-15 Ultracel-10k (Millipore). A final purified sample ofsoluble cynomolgus monkey IL-6 receptor (hereinafter cIL-6R) wasobtained through further purification on a Superdex200pg16/60 gelfiltration column (GE Healthcare Bioscience).

Preparation of Recombinant Cynomolgus Monkey IL-6 (cIL-6)

Cynomolgus monkey IL-6 was prepared by the procedure described below.The nucleotide sequence encoding 212 amino acids deposited underSWISSPROT Accession No. P79341 was prepared and cloned into a mammaliancell expression vector. The resulting vector was introduced into CHOcells to prepare a stable expression cell line (cyno.IL-6-producing CHOcell line). The culture medium of cyno.IL-6-producing CHO cells waspurified using a SP-Sepharose/FF column (GE Healthcare Bioscience) andthen concentrated with Amicon Ultra-15 Ultracel-5k (Millipore). A finalpurified sample of cynomolgus monkey IL-6 (hereinafter cIL-6) wasobtained through further purification on a Superdex75pg26/60 gelfiltration column (GE Healthcare Bioscience), followed by concentrationwith Amicon Ultra-15 Ultracel-5k (Millipore).

Preparation of a Known High-Affinity Anti-IL-6 Receptor Antibody

A mammalian cell expression vector was constructed to express VQ8F11-21hIgG1, a known high-affinity anti-IL-6 receptor antibody. VQ8F11-21hIgG1 is described in US 2007/0280945 A1 (US 2007/0280945 A1; the aminoacid sequences of H chain and L chain as set forth in SEQ ID NOs: 77 and78, respectively). The antibody variable region was constructed by PCRusing a combination of synthetic oligo DNAs (assembly PCR) and IgG1 wasused for the constant region. The antibody variable and constant regionswere combined together by assembly PCR, and then inserted into amammalian expression vector to construct expression vectors for the Hchain and L chain of interest. The nucleotide sequences of the resultingexpression vectors were determined by a method known to those skilled inthe art. The high-affinity anti-IL-6 receptor antibody (hereinafterabbreviated as “control”) was expressed and purified using theconstructed expression vectors by the method described in Example 1.

Preparation, Expression, and Purification of TOCILIZUMAB Variants

TOCILIZUMAB variants were prepared using the QuikChange Site-DirectedMutagenesis Kit (Stratagene) according to the method described in theappended instruction manual. The resulting plasmid fragments wereinserted into mammalian cell expression vectors to construct expressionvectors for the H chains and L chains of interest. The nucleotidesequences of the obtained expression vectors were determined by a methodknown to skilled artisans. The antibodies were expressed by the methoddescribed below. Human embryonic kidney cancer-derived HEK293H cell line(Invitrogen) was suspended in DMEM (Invitrogen) supplemented with 10%Fetal Bovine Serum (Invitrogen). The cells were plated at 10 ml per dishin dishes for adherent cells (10 cm in diameter; CORNING) at a celldensity of 5 to 6×10⁵ cells/ml and cultured in a CO₂ incubator (37° C.,5% CO₂) for one whole day and night. Then, the medium was removed byaspiration, and 6.9 ml of CHO-S-SFM-II medium (Invitrogen) was added.The prepared plasmid was introduced into the cells by the lipofectionmethod. The resulting culture supernatants were collected, centrifuged(approximately 2000 g, 5 min, room temperature) to remove cells, andsterilized by filtering through 0.22-μm filter MILLEX(R)-GV (Millipore)to obtain the supernatants. Antibodies were purified from the obtainedculture supernatants by a method known to those skilled in the art usingrProtein A Sepharose™ Fast Flow (Amersham Biosciences). To determine theconcentration of the purified antibody, absorbance was measured at 280nm using a spectrophotometer. Antibody concentrations were calculatedfrom the determined values using an absorbance coefficient calculated bythe PACE method (Protein Science 1995; 4:2411-2423).

Establishment of a Human gp130-Expressing BaF3 Cell Line

A BaF3 cell line expressing human gp130 was established by the proceduredescribed below to obtain a cell line that proliferates in anIL-6-dependent manner.

A full-length human gp130 cDNA (Hibi et al., Cell (1990) 63:1149-1157(GenBank #NM_(—)002184)) was amplified by PCR and cloned into theexpression vector pCOS2Zeo to construct pCOS2Zeo/gp130. pCOS2Zeo is anexpression vector constructed by removing the DHFR gene expressionregion from pCHOI (Hirata et al., FEBS Letter (1994) 356:244-248) andinserting the expression region of the Zeocin resistance gene. Thefull-length human IL-6R cDNA was amplified by PCR and cloned intopcDNA3.1(+) (Invitrogen) to construct hIL-6R/pcDNA3.1(+).

10 μg of pCOS2Zeo/gp130 was mixed with BaF3 cells (0.8×10⁷ cells)suspended in PBS, and then pulsed at 0.33 kV and 950 μFD using GenePulser (Bio-Rad). The BaF3 cells having the gene introduced byelectroporation were cultured for one whole day and night in RPMI 1640medium (Invitrogen) supplemented with 0.2 ng/ml mouse interleukin-3(Peprotech) and 10% fetal bovine serum (hereinafter FBS, HyClone), andselected by adding RPMI 1640 medium supplemented with 100 ng/ml humaninterleukin-6 (R&D systems), 100 ng/ml human interleukin-6 solublereceptor (R&D systems), and 10% FBS to establish a humangp130-expressing BaF3 cell line (hereinafter “BaF3/gp130”). ThisBaF/gp130 proliferates in the presence of human interleukin-6 (R&Dsystems) and soluble human IL-6 receptor, and thus can be used to assessthe growth inhibition activity (or IL-6 receptor neutralizing activity)of an anti-IL-6 receptor antibody.

Assessment for the Bbiological Activity by Human gp130-Expressing BaF3Cells (BaF/gp130)

The IL-6 receptor neutralizing activity was assessed using BaF3/gp130which proliferates in an IL-6/IL-6 receptor-dependent manner. Afterthree washes with RPMI1640 supplemented with 10% FBS, BaF3/gp130 cellswere suspended at 5×10⁴ cells/ml in RPMI1640 supplemented with 600 ng/mlor 60 ng/ml human interleukin-6 (TORAY) (final concentration of 300ng/ml or 30 ng/ml), appropriate amount of soluble human IL-6 receptor,and 10% FBS. The cell suspensions were dispensed (50 μl/well) into96-well plates (CORNING). Then, the purified antibodies were dilutedwith RPMI1640 containing 10% FBS, and added to each well (50 μl/well).The cells were cultured at 37° C. under 5% CO₂ for three days. WST-8Reagent (Cell Counting Kit-8; Dojindo Laboratories) was diluted two-foldwith PBS. Immediately after 20 μl of the reagent was added to each well,the absorbance at 450 nm (reference wavelength: 620 nm) was measuredusing SUNRISE CLASSIC (TECAN). After culturing for two hours, theabsorbance at 450 nm (reference wavelength: 620 nm) was measured again.The IL-6 receptor neutralizing activity was assessed using the change ofabsorbance during two hours as an indicator.

Biacore-Based Analysis of Binding to Soluble Human IL-6 Receptor

Antigen-antibody reaction kinetics was analyzed using Biacore T100 (GEHealthcare). The soluble human IL-6 receptor-antibody interaction wasmeasured by immobilizing appropriate amounts of protein A or protein A/Gor anti-IgG (γ-chain specific) F(ab′)₂ onto a sensor chip by aminecoupling method, binding antibodies of interest onto the chip at pH7.4,and then running soluble IL-6 receptor adjusted to variousconcentrations at pH7.4 over the chip as an analyte. All measurementswere carried out at 37° C. The kinetic parameters, association rateconstant k_(a) (1/Ms) and dissociation rate constant k_(d) (1/s) werecalculated from the sensorgrams obtained by measurement. Then, K_(D) (M)was determined based on the rate constants. The respective parameterswere determined using Biacore T100 Evaluation Software (GE Healthcare).

Assessment for the pH-Dependent Dissociation from Membrane-Type IL-6Receptor using Biacore

The antigen-antibody reaction with membrane-type IL-6 receptor at pH 5.8and pH 7.4 was observed using Biacore T100 (GE Healthcare). The bindingto membrane-type IL-6 receptor was assessed by evaluating the binding tosoluble human IL-6 receptor immobilized onto the sensor chip. SR344 wasbiotinylated by a method known to those skilled in the art. Based on theaffinity between biotin and streptavidin, biotinylated soluble humanIL-6 receptor was immobilized onto the sensor chip via streptavidin. Allmeasurements were conducted at 37° C. The mobile phase buffer was 10 mMMES (pH 5.8), 150 mM NaCl, and 0.05% Tween 20. A clone exhibitingpH-dependent binding was injected under the condition of pH 7.4 to bindto soluble human IL-6 receptor (injection sample buffer was 10 mM MES(pH 7.4), 150 mM NaCl, and 0.05% Tween 20). Then, the pH-dependentdissociation of each clone was observed at pH 5.8, which is the pH ofthe mobile phase. The dissociation rate constant (kd (1/s)) at pH 5.8was calculated using Biacore T100 Evaluation Software (GE Healthcare) byfitting only the dissociation phase at pH 5.8. The sample concentrationwas 0.25 μg/ml. Binding was carried out in 10 mM MES (pH 7.4), 150 mMNaCl, and 0.05% Tween 20, and dissociation was carried out in 10 mM MES(pH 5.8), 150 mM NaCl, and 0.05% Tween 20. Likewise, the dissociationrate constant (kd (1/s)) at pH 7.4 was calculated using Biacore T100Evaluation Software (GE Healthcare) by fitting only the dissociationphase at pH 7.4. The sample concentration was 0.5 μg/ml. Binding wascarried out in 10 mM MES (pH 7.4), 150 mM NaCl, and 0.05% Tween 20, anddissociation was carried out in 10 mM MES (pH 7.4), 150 mM NaCl, and0.05% Tween 20.

Assessment of the Binding to Human FcRn

FcRn is a complex of FcRn and β2-microglobulin. Oligo-DNA primers wereprepared based on the human FcRn gene sequence disclosed (J. Exp. Med.(1994) 180(6):2377-2381). A DNA fragment encoding the whole gene wasprepared by PCR using human cDNA (Human Placenta Marathon-Ready cDNA,Clontech) as a template and the prepared primers. Using the obtained DNAfragment as a template, a DNA fragment encoding the extracellular domaincontaining the signal region (Met1-Leu290) was amplified by PCR, andinserted into a mammalian cell expression vector (the amino acidsequence of human FcRn as set forth in SEQ ID NO: 79). Likewise,oligo-DNA primers were prepared based on the human β2-microglobulin genesequence disclosed (Proc. Natl. Acad. Sci. USA. (2002)99(26):16899-16903). A DNA fragment encoding the whole gene was preparedby PCR using human cDNA (Hu-Placenta Marathon-Ready cDNA, CLONTECH) as atemplate and the prepared primers. Using the obtained DNA fragment as atemplate, a DNA fragment encoding the whole β2-microglobulin containingthe signal region (Met1-Met119) was amplified by PCR and inserted into amammalian cell expression vector (the amino acid sequence of humanβ2-microglobulin as set forth in SEQ ID NO: 80).

Soluble human FcRn was expressed by the following procedure. Theplasmids constructed for human FcRn and β2-microglobulin were introducedinto cells of the human embryonic kidney cancer-derived cell lineHEK293H (Invitrogen) using 10% FBS (Invitrogen) by lipofection. Theresulting culture supernatant was collected, and FcRn was purified usingIgG Sepharose 6 Fast Flow (Amersham Biosciences) by the method describedin J. Immunol. 2002 Nov. 1; 169(9):5171-80, followed by furtherpurification using HiTrap Q HP (GE Healthcare).

Determination of Antibody Concentration in Mouse Plasma

Antibody concentrations in mouse plasma were determined by ELISAaccording to a method known to those skilled in the art.

PK/PD Test to Determine the Antibody Concentration in the Plasma, CRPConcentration, and free Soluble IL-6 Receptor in Monkeys

The plasma concentrations in cynomolgus monkeys were determined by ELISAusing a method known to those skilled in the art.

The concentration of CRP was determined with an automated analyzer(TBA-120FR; Toshiba Medical Systems Co.) using Cias R CRP (KANTOCHEMICAL CO., INC.).

The plasma concentration of free soluble cynomolgus monkey IL-6 receptorin cynomolgus monkeys was determined by the procedure described below.All IgG-type antibodies (cynomolgus monkey IgG, anti-human IL-6 receptorantibody, and anti-human IL-6 receptor antibody-soluble cynomolgusmonkey IL-6 receptor complex) in the plasma were adsorbed onto Protein Aby loading 30 μl of cynomolgus monkey plasma onto an appropriate amountof rProtein A Sepharose Fast Flow resin (GE Healthcare) dried in a0.22-μm filter cup (Millipore). Then, the solution in cup was spinneddown using a high-speed centrifuge to collect the solution that passedthrough. The solution that passed through does not contain ProteinA-bound anti-human IL-6 receptor antibody-soluble cynomolgus monkey IL-6receptor complex. Therefore, the concentration of free soluble IL-6receptor can be determined by measuring the concentration of solublecynomolgus monkey IL-6 receptor in the solution that passed throughProtein A. The concentration of soluble cynomolgus monkey IL-6 receptorwas determined using a method known to those skilled in the art formeasuring the concentrations of soluble human IL-6 receptor. Solublecynomolgus monkey IL-6 receptor (cIL-6R) prepared as described above wasused as a standard. The percentage of free soluble IL-6 receptor wascalculated by the following formula.

$\frac{\mspace{14mu}\begin{matrix}{{{Free}\mspace{14mu}{soluble}\mspace{14mu}{IL}\text{-}6\mspace{14mu}{receptor}\mspace{14mu}{concentration}}\mspace{14mu}} \\{{after}\mspace{14mu}{antibody}\mspace{14mu}{administration}}\end{matrix}}{\;\begin{matrix}{{{Soluble}\mspace{14mu}{IL}\text{-}6\mspace{14mu}{receptor}\mspace{14mu}{concentration}}\mspace{11mu}} \\{{before}\mspace{14mu}{antibody}\mspace{14mu}{administration}}\end{matrix}} \times 100$

The invention claimed is:
 1. An isolated anti-IL-6 receptor antibodythat comprises a heavy chain variable region comprising the sequence ofSEQ ID NO: 20 and a light chain variable region comprising the sequenceof SEQ ID NO:
 23. 2. A pharmaceutical composition comprising theantibody of claim
 1. 3. An isolated anti-IL-6 receptor antibody thatcomprises a heavy chain comprising the sequence of SEQ ID NO: 26 and alight chain comprising the sequence of SEQ ID NO:
 29. 4. Apharmaceutical composition comprising the antibody of claim
 3. 5. Anisolated nucleic acid encoding an anti-IL-6 receptor antibody thatcomprises a heavy chain variable region comprising the sequence of SEQID NO: 20 and a light chain variable region comprising the sequence ofSEQ ID NO:
 23. 6. A vector comprising the nucleic acid of claim
 5. 7. Anisolated host cell comprising the vector of claim
 6. 8. A method forproducing an antibody that comprises a heavy chain variable regioncomprising the sequence of SEQ ID NO: 20 and a light chain variableregion comprising the sequence of SEQ ID NO: 23, the method comprisingculturing the host cell of claim 7 under conditions suitable to producethe antibody.
 9. An isolated nucleic acid encoding an anti-IL-6 receptorantibody that comprises a heavy chain comprising the sequence of SEQ IDNO: 26 and a light chain comprising the sequence of SEQ ID NO:
 29. 10. Avector comprising the nucleic acid of claim
 9. 11. An isolated host cellcomprising the vector of claim
 10. 12. A method for producing anantibody that comprises a heavy chain variable region comprising thesequence of SEQ ID NO: 26 and a light chain variable region comprisingthe sequence of SEQ ID NO: 29, the method comprising culturing the hostcell of claim 11 under conditions suitable to produce the antibody. 13.An isolated nucleic acid comprising a sequence encoding a polypeptidecomprising the sequence of SEQ ID NO:
 20. 14. A vector comprising thenucleic acid of claim
 13. 15. An isolated nucleic acid comprising asequence encoding a polypeptide comprising the sequence of SEQ ID NO:23.
 16. A vector comprising the nucleic acid of claim
 15. 17. Anisolated host cell containing (i) a vector comprising a nucleic acidencoding a polypeptide comprising SEQ ID NO: 20 and (ii) a vectorcomprising a nucleic acid encoding a polypeptide comprising the sequenceof SEQ ID NO:
 23. 18. A method for producing an antibody that comprisesa heavy chain variable region comprising the sequence of SEQ ID NO: 20and a light chain variable region comprising the sequence of SEQ ID NO:23, the method comprising culturing the host cell of claim 17 underconditions suitable to produce the antibody.
 19. The method of claim 18,wherein the antibody comprises a heavy chain comprising the sequence ofSEQ ID NO: 26 and a light chain comprising the sequence of SEQ ID NO:29.